Nanostructures from Laser-Ablated Nanohole Templates

ABSTRACT

Solution casting a nanostructure. Preparing a template by ablating nanoholes in a substrate using single-femtosecond laser machining. Replicating the nanoholes by applying a solution of a polymer and a solvent into the template. After the solvent has substantially dissipated, removing the replica from the substrate.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/599,926, filed Feb. 16, 2012, and entitled “Ultra-High Aspect RatioNanostructure from Laser Ablated Nanoholes;” the disclosure of which ishereby incorporated herein in its entirety.

TECHNICAL FIELD

The present disclosure relates generally to nanotechnology. Embodimentsof the disclosed technology relate more specifically to nanostructuresmade using templates created using single pulse femtosecond lasermachining

BACKGROUND

Nanotechnology, in general, encompasses preparation, manipulation, andapplications of nanoscale materials and structures. One reason behindgoing down in length scale is the deviation observed in materialproperties (physical, chemical, and mechanical) due to the manifestationof the statistical-mechanical and quantum-mechanical effects, which areotherwise not observed in their bulk counterparts. For example, theelectronic properties of a material can significantly alter with thereduction in the size scale of the material because of pronouncedquantum mechanical effects, which are more dominant when less than 100nm size scale is achieved. Indeed, the terminology nano, from amaterials perspective, is now typically applied to materials with atleast one dimension less than 100 nm so that the quantum size effect canbe observed and exploited for practical purposes.

Nanostructures are nanoscale materials with at least one dimension lessthan 100 nm per current scientific definition. Although this definitionof nanostructures/nanomaterials is now widely accepted, it is still verycommon to regard anything on sub-micron (<1 μm) length scale as nano forsimplicity on a case-to-case basis. Nanostructures or nanomaterials areconsidered to be the basis of nanotechnology; other aspects beingnanofabrication, nanoscale characterization, and nanodevice systems.Nanofabrication is a collection of fabrication strategies used for thepreparation of nanomaterials or nanostructures, and is broadlyclassified into top-down and bottom-up techniques. Nanoscalecharacterization is the examination of properties of nanoscale materialswith the use of highly sophisticated analytical instruments, whereasnanodevice systems are mission-oriented nanoscale machines or apparatusmade from several nanoscale objects or just molecules.Nanoscale-diameter-holes are referred to as nanoholes.

One of the most common bottom-up nanofabrication techniques used for thepreparation of nanostructures is template synthesis. It has been used toprepare one-dimensional (1D) nanostructures from a variety of materials.Based on their morphology, nanostructures may be classified asnanowires, nanoribbons, nanopyramids, nanopillars, and nanocones, andmay be synthesized either as topographical features on films orsubstrates via nanofabrication techniques or by solution synthesis.

Template synthesis may be described as a casting process, in which amold is subjected to a deposition process to create structures withinthe holes/pores of the mold. The morphology and dimensions of theresulting structures depend on that of the holes in the mold from whichthey are cast. The four generic casting methods employed in templatesynthesis are: soft lithography, electrochemical or electrolessdeposition, vapor deposition, and injection molding.

Although there are several different types of molds used in the templatesynthesis of nanostructures, the four most commonly used are: anodizedaluminum oxide (AAO); nanochannel glass (NCG); ion track-etch templates;and lithographically patterned templates (referred to as master patternin soft lithography).

A variety of materials, e.g., metal, semiconductor, insulator,superconductor, and polymer, have been imprinted by template synthesisusing AAO, NCG, and track-etch substrates. Attention has been paid toboth the template preparation and imprinting strategies, so that highquality nano/micro structures can be fabricated and used. Although thesetemplates have been useful for numerous applications, there still aremany applications where they cannot fulfill the requirements. Onedisadvantage of these templates is the inability to control the physicalarrangement of the nanoholes. Furthermore, in case of AAO and track-etchbased template synthesis, the template is eventually dissolved in achemical solution to harvest the cast structure. In summary, reusabletemplates with user-defined arrangement of nanoholes cannot befabricated by any of these three techniques. By comparison,nanolithography offers ever-improving control on the physicalarrangement of the nanoholes that a user can pattern on rigidsubstrates.

Nanolithography is essentially a patterning technique in whichstructures (e.g., arrays of nanoholes) of user-defined designs areproduced at the surface of substrates such as silicon, fused silica, andacrylic glass. The lateral and vertical dimensions of the nanoholes areusually controlled post-patterning by an etching treatment (to increasethe hole size) or a deposition treatment (to decrease the hole size bycoating its sidewalls). Nanolithography constitutes a set of lithographytechniques of which most routinely used are photolithography, electronbeam lithography, and soft lithography.

Photolithography is a multi-step lithography process in whichuser-defined patterns may be transferred to substrates with the help ofultraviolet light and a pattern-carrying mask. In this process, thesubstrate first may be cleaned with wet chemical treatments to renderthe surface free of organic and inorganic impurities. The substrate thenmay be coated with a layer of photoresist by spin coating after exposingthe substrate to a chemical that improves adhesion between thephotoresist and the substrate. The photoresist coated substrate then maybe heated at 80° C.-100° C. on a hot plate for up to a minute to expelexcess photoresist solvent from the photoresist layer in a processcalled pre-baking, so that the substrate is ready for UV exposure. Thephotoresist then may be exposed to UV light through a photomask carryingthe pattern, and then subjected to another baking process calledpost-exposure baking. A photomask is essentially a fused silica platewith user-defined patterns made on chromium. The UV light exposedphotoresist may be chemically modified, which develops patterns onto thephotoresist. Two types of photoresists exist: positive photoresist andnegative photoresist. The UV exposed region on a positive photoresist iswashed out upon developing, whereas the opposite results in case of anegative photoresist, i.e., the unexposed region is washed out. Thepenultimate step in photolithography is the pattern transfer process inwhich the patterned photoresist may be subjected to an etching treatment(dry or wet) for a time period corresponding to the desired featuresize, which removes parts of the substrate not covered with thephotoresist. Dry etching is an anisotropic etching process, whichprimarily may be employed when high aspect ratio and deep features aredesired. The most common of all the dry etching processes is deepreactive-ion etching (DRIE) based on the Bosch process in which severalhundred microns deep features may be produced on the substrate. Incomparison, wet chemical etching is an isotropic etching process inwhich the maximum attainable depth for a feature equals the thickness ofthe photoresist used. The final step is the removal of the leftoverphotoresist. The resolution of the feature that may be produced on thesubstrate depends on the wavelength of the UV light used to expose thephotoresist; in principle, the shorter the wavelength, the smaller thefeature that may be produced. Thus, high-resolution features can beproduced by reducing the wavelength. Over the years, many differenttypes of UV sources have been used starting from gas-discharge lampswith up to 365 nm wavelength (till mid-1980s) to excimer lasers with 193nm (current technology), which is expected to be superseded by theextreme ultra violet (EUV) source with up to 13.5 nm (also termed asnext generation lithography. However, the depth of focus is negativelyaffected by the wavelength of the UV light, which restricts thethickness of the photoresist and eventually the depth of the finalpattern.

EBL is a maskless lithography technique by which complex features may beproduced on a substrate with very high resolution. The operationalprinciple of EBL is similar to that of photolithography with theexception that EBL is a direct-write process where patterns may bedirectly engineered onto the substrate without the need of a mask. Thesubstrate may be coated with a thin layer of resist (e.g.,polymethylmethacrylate) by spin coating, pre-baked, subjected to patternwriting in an electron beam lithography system (e-beam system), followedby resist development and pattern transfer. One advantage of EBL overphotolithography is that it is not diffraction-limited, and featureswith resolutions of up to 20 nm may be produced; indeed, sub-10 nmfeatures can also be produced by EBL. However, the resolution of EBL islimited by the scattering of electrons in the resist. Furthermore, thethroughput of EBL is very low as the processing time is directlyproportional to the pattern area for a certain dose given by theequation T*I=D*A, where T is the exposure time, I is the beam current, Dis the dose in Coulombs/cm², and A is the exposed area. In other words,it would take approximately 12 days to pattern a 1 cm² area with a 1 nAbeam current and 1 mC/cm² dose.

Soft lithography is a form of nanolithography constituting a set ofvarious non-photolithographic techniques centered on the principle ofself-assembly and replication (or imprinting). The term “soft” in softlithography is derived from the physical nature of the mold used (i.e.,soft) for fabricating structures, where the soft mold itself is preparedfrom rigid templates. Rigid templates, also termed as master patterns,are reusable patterned substrates that are usually prepared byphotolithography, EBL, or micromachining on materials like silicon andsilica. The master pattern is first subjected to cast molding to formits replica on an elastomeric material like polydimethylsiloxane (PDMS)to prepare the soft mold or stamp, which is then used to generatestructures or patterns by various techniques that are collectivelytermed as soft lithography. Over the years, soft lithography has evolvedinto one of the most economical and most convenient nanolithographytechniques where features as small as sub-10 nm size scale have beensuccessfully fabricated. Some commonly used soft lithography techniquesare: replica molding (REM), microtransfer molding (μTM), micromolding incapillaries (MIMIC), solvent-assisted micromolding (SAMIM), andmicrocontact printing (μCP).

REM is a single-step replication technique used to createmicro/nanoscale features by curing prepolymers cast into PDMS molds. UVor thermally curable prepolymers without any solvent and low shrinkage(typically less than 3% on curing) are used for REM. Polyurethane (PU)is an example of a UV curable prepolymer used extensively for replicamolding. REM has also been employed against rigid molds for massproduction of a variety of materials including compact discs (CDs),diffraction gratings, holograms, and micro-tools.

In a μTM process, a liquid prepolymer is first applied to the surface ofa PDMS mold and the excess is removed by scraping, or blowing by passinga stream of gas, and then the liquid prepolymer filled mold is placed onthe surface of a substrate. The prepolymer is thermally or UV cured tobecome solid and attached to the substrate after which the PDMS mold isdelicately removed to leave behind the patterned structure on thesubstrate. Thermally curable epoxy resins and UV curable polyurethaneshave been used as prepolymers for μTM.

MIMIC is a soft lithography method used to create micro/nanoscalefeatures from a variety of materials, such as UV or thermally curablepolymers with no solvents, functional polymer solutions, glassy carbonor ceramic forming precursor polymers, inorganic salts, sol-gelmaterials, colloidal solutions, polymer beads, and biologicallyfunctional macromolecules. In this method, a PDMS mold with channels isfirst placed on a substrate and then a low-viscosity prepolymer isplaced at the open ends of the channels. The prepolymer fills up thechannels by capillary action, which is then solidified by curing. ThePDMS mold is then slowly removed from the substrate to create patternedfeatures of the polymer.

SAMIM utilizes polymer films to create nanostructures on their surfacefrom PDMS molds. However, only those polymer films can be used whosesolvents do not affect the PDMS mold during micromolding. In thismethod, the surface of the PDMS mold is first wetted with a solvent thatcan dissolve (or soften) the polymer whose nanostructures are to becreated. The PDMS mold with the solvent is then brought in contact withthe polymer film held on a substrate. The solvent dissolves (or softens)a thin layer of the polymer and forms a gel that fills the patterns inthe mold. When the solvent evaporates the gel inside the patternsfreezes into solid polymer and forms polymer nanostructures on thesubstrate.

μCP is a printing process in which a soft mold like PDMS is covered witha chemical solution called ink and then brought into physical contactwith a substrate after the ink has dried. A commonly used ink is a thiolsolution, such as hexadecanethiol. Upon physical contact with thesubstrate, the thiol is transferred to the surface of the substrateresulting in the reproduction of the mold pattern. The three knownconfigurations to transfer the ink, based on the geometry of the moldand the substrate, are: planar mold onto a planar substrate, nonplanarmold onto a planar substrate, and planar mold onto a nonplanarsubstrate. A derivative of μCP called nanocontact printing (NCP) isreported to pattern sub-50 nm features. μCP (and NCP) have beeninvestigated for applications in micromachining (where the ink acts as aresist for an etching process) and biological engineering (by patterningcells, DNA, and proteins).

In NIL, an imprint resist is first spin coated on the surface of asubstrate and then pressed against a rigid mold whose features are to bereproduced. Once the right pressure is achieved between the imprintresist and the template, the imprint resist is either subjected to athermal treatment or a UV light treatment for curing depending on thetype of resist. If a thermoplastic polymer is chosen as the imprintresist, it is heated above its glass transition temperature to softenand acquire the impression of the template. The template is then slowlyseparated from the imprint upon cooling. The acquired impression in theimprint is the negative replica of the template, which is furthersubjected to a pattern transfer process (usually by reactive ion etchingjust like photolithography) to transmit the imprinted pattern in theresist to the substrate.

Very similar to this technique is the photo nanoimprint lithography(P-NIL), also called cold embossing or UV nanoimprint lithography(UV-NIL), in which a photoresist is used as an imprint material. Thephotoresist is either spin coated or dispensed with a scanning inkjethead (step and flash imprint lithography) on a transparent substrate,brought in contact with the template to maintain a certain pressure inbetween, and then cured by exposing to UV light. The photoresistundergoes crosslinking and hardens, and acquires the impression of thetemplate after which they are slowly detached from each other. This stepis also followed by a pattern transfer process to transmit the featuresfrom the imprint into the substrate.

Developed as a low manufacturing cost and high-throughput method tofabricate nanoscale features, the NIL technique was demonstrated tocreate features from 25-nm resolution up to sub-10 nm resolution, andwas successfully applied to produce magnetic nanostructures, quantizedmagnetic disks, silicon field effect transistors, semiconductornanowires, and nano-compact disks. Indeed, it was shown that NIL can bescaled up to fabricate large area patterns as well.

Many more techniques have been investigated and reported for patterningsubstrates, such as laser based nanolithography techniques includingfemtosecond laser micromachining, dip pen lithography (DPN), extreme UVlithography (EUV), proximal-probe lithography, x-ray lithography, ionbeam lithography including focused ion beam milling, proton beamwriting, and more recently neutral particle lithography.

SUMMARY

The present technology includes methods for preparing nanostructures andthe products made by those methods. In some embodiments, a template canbe prepared by ablating nanoholes in a substrate usingsingle-femtosecond laser machining. The nanoholes can be replicated byapplying a solution of a polymer and a solvent into the template. Afterthe solvent has substantially dissipated, the replica can be removedfrom the substrate. In some embodiments, the polymer solution comprisesone of: cellulose acetate in acetone, polycaprolactone (PCL) inchloroform, PCL-polyethylene glycol in chloroform, polydimethylsiloxanein heptane, polymethylmethacrylate in toluene, polyvinyl alcohol inde-ionized water, and collodion in amyl acetate. In some embodiments,the polymer is capable of forming a continuous film. In some suchcontinuous film embodiments, the polymer is capable of forming acontinuous film through the application of external energy. In someembodiments, the polymer solution comprises a two percent (2%) solutionby weight of one of: cellulose acetate in acetone, polycaprolactone(PCL) in chloroform, PCL-polyethylene glycol in chloroform, andcollodion in amyl acetate; while in other embodiments, the polymersolution comprises a solution between two percent (2%) and ten percent(10%) by weight of cellulose acetate in acetone. In yet furtherembodiments, the polymer solution comprises a twenty five percent (25%)solution by weight of polydimethylsiloxane in heptane; or a solution ofbetween five percent (5%) and ten percent (10%) by weight ofpolymethylmethacrylate in toluene; or a five percent (5%) solution byweight of polyvinyl alcohol in de-ionized water.

In some embodiments, prior to applying a solution of a polymer and asolvent into the template, a fluorocarbon-based antistick coating can beapplied to the template. In some such embodiments, thefluorocarbon-based antistick coating is prepared fromperfluorodecyltrichlorosilane.

In some embodiments, a template can be prepared by ablating nanoholes ina substrate using single-femtosecond laser machining. The nanoholes canbe replicated by applying a polymer resin into the template. Afterallowing the resin to set, the replica can be removed from thesubstrate.

In some embodiments, a template can be prepared by ablating nanoholes ina substrate using single-femtosecond laser machining. The nanoholes canbe replicated by casting a solution of a polyethylene and a solvent intothe template. After the solvent has been allowed to substantiallydissipate, the polyethylene as cast in the template can be meltedthrough application of energy. After allowing the melted polyethylene tocool, the cooled polyethylene replica can be removed from the substrate.In some such embodiments, the polymer solution comprises ten percent(10%) solution by weight of polyethylene in toluene. In some suchembodiments, melting comprises heating to about one hundred fifty five(155) degrees Celsius for about two (2) minutes; and cooling comprisescooling at room temperature for at least about two (2) hours.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents Scanning Electron Microscope (SEM) images of nanoholesproduced at the surface of fused silica.

FIG. 2 presents SEM images of the replicas of femtosecond laser machinedfused silica shown in FIG. 1.

FIG. 3 is an SEM image of nanoholes.

FIG. 4 is a schematic of an amplified femtosecond laser pulsesgeneration in an amplified femtosecond system.

FIG. 5 illustrates a femtosecond laser beam propagation path.

FIG. 6 illustrates two lenses of a beam expander.

FIG. 7 illustrates the optical assembly of a workstation used for lasermachining of fused silica.

FIG. 8 illustrates surface topography of a substrate: fine scan (A) andinterpolation of the fine scan (B).

FIG. 9 illustrates preparation of cellulose acetate nanowires bynanoimprinting from a single-pulse femtosecond laser nanomachined fusedsilica template using a 35 μm cellulose acetate film.

FIG. 10 illustrates preparation of polymer solution nanowires bynanoimprinting from a single-pulse femtosecond laser nanomachined fusedsilica template by solution casting.

FIG. 11 illustrates a silica deposition process for functionalization ofnanowires.

FIG. 12 illustrates a 2D gradient pattern (reduced density for visualclarity and not to scale) fabricated on a 500 μm thick fused silicasubstrate by single-pulse femtosecond laser machining

FIG. 13 is a schematic of silica nanoneedle preparation for cell studiesusing National Institutes of Health (NIH) 3T3 mouse embryo fibroblasts.

FIG. 14 is a schematic of the z-stage movement during femtosecond lasermachining of fused silica.

FIG. 15 is a SEM image of nanoholes made at the surface of fused silica.

FIG. 16 is a SEM image of the cellulose acetate replica imprinted fromnanoholes (taken at 45° tilt) shown in FIG. 15.

FIG. 17 is a schematic of SEM imaging of a cellulose acetate imprint ofa nanohole showing its projected length and the actual length.

FIG. 18 illustrates the dependence of hole diameter on the position ofoptical focus with respect to a substrate surface.

FIG. 19 illustrates the dependence of hole depth on the position ofoptical focus with respect to the substrate surface.

FIG. 20 is a SEM image of PCL nanowires imprinted from an as-machinedfused silica template using 2 wt % PCL solution in chloroform.

FIG. 21 is a SEM image of polycaprolactone micropillars imprinted froman etched fused silica template using 2 wt % polycaprolactone solutionin chloroform.

FIG. 22 is a SEM image of PCL-PEG nanowires imprinted from anas-machined fused silica template using 2 wt % polycaprolactone solutionin chloroform.

FIG. 23 is a SEM image of PCL-PEG micropillars imprinted from an etchedfused silica template using 2 wt % PCL-PEG solution in chloroform.

FIG. 24 is a SEM image of cellulose acetate nanowires (nanocones)imprinted from an as-machined fused silica template using 5 wt %cellulose acetate solution in acetone.

FIG. 25 is a SEM image of cellulose acetate micropillars imprinted froman etched fused silica template using 5 wt % cellulose acetate solutionin acetone.

FIG. 26 is a SEM image of polyethylene nanowires (nanocones) imprintedfrom an as-machined fused silica template using 10 wt % polyethylenesolution in toluene.

FIG. 27 is a SEM image of polyethylene micropillars imprinted from anetched fused silica template using 10 wt % polyethylene solution intoluene.

FIG. 28 is a SEM image of PVA nanowires imprinted from an as-machinedfused silica template using 5 wt % PVA solution in DI water.

FIG. 29 is a SEM image of PVA micropillars imprinted from an etchedfused silica template using 5 wt % PVA solution in DI water.

FIG. 30 is a SEM image of PMMA nanowires imprinted from an as-machinedfused silica template using 10 wt % PMMA solution in toluene.

FIG. 31 is a SEM image of PMMA micropillars imprinted from an etchedfused silica template using 10 wt % PMMA solution in toluene.

FIG. 32 is a SEM image of Collodion nanowires imprinted from anas-machined fused silica template using a 2% Collodion solution in amylacetate.

FIG. 33 is a SEM image of Collodion micropillars imprinted from anetched fused silica template using 2% Collodion solution in amylacetate.

FIG. 34 is a SEM image of PDMS nanocones imprinted from an as-machinedfused silica template using 33 wt % PDMS solution in n-heptane.

FIG. 35 is a SEM image of PDMS micropillars imprinted from an etchedfused silica template using 33 wt % PDMS solution in n-heptane.

FIG. 36 illustrates the rate of deposition of silica as a function ofexposure time in a first beaker in accordance with functionalization ofnanostructures of the current technology.

FIG. 37 is an SEM image of an array of silica nanowires obtained bycoating cellulose acetate nanowires with silica for 5 minutes in thefirst beaker in accordance with functionalization of nanostructures ofthe current technology.

FIG. 38 is an EDS spectrum of the tip of a silica nanowire shown on topright.

FIG. 39 illustrates the absorption spectrum of a silica coating recordedon an ATR-FTIR.

FIG. 40 present SEM images of a silica nanowire subjected to gallium ionsectioning in an SEM/FIB. Each image represents a different sectionfurther down the silica nanocone from (A) to (D). The final image (D)shows an open-tip silica nanoneedle.

FIG. 41 is an SEM image of nanoholes from an edge of the 2D gradientpattern.

FIG. 42 is an SEM image of silica nanoneedles made by coating celluloseacetate nanowires with silica.

FIG. 43 presents SEM images of cells fixed on flat silica (A) and silicananoneedles (B).

FIG. 44 illustrates a comparison between cellular response to flatsilica and silica nanoneedles.

FIG. 45 illustrates how cell behavior can be influenced by the presenceand spacing of silica nanoneedles.

FIG. 46 presents SEM images of nanoholes made at the surface of fusedsilica by aspheric lens machining at different positions normal to thesurface of the fused silica.

FIG. 47 presents SEM images of cellulose acetate replica of nanoholesshown in FIG. 46.

FIG. 48 are schematic representations of the single pulse femtosecondlaser machining configurations modeled.

FIG. 49 illustrates the spot radius at the image plane after the laserbeam focused through the aspheric lens.

FIG. 50 illustrates the surface contour of the fast Fourier transform(FFT) of the Huygens point spread function (PSF) at the image planecorresponding to FIG. 49.

FIG. 51 illustrates a beam path originating from an amplifiedfemtosecond laser set-up (bottom left), passing through the zoom lens,and focusing on the substrate (mounted on a motorized stage) through theaspheric lens used for single pulse femtosecond laser machining with anaspheric lens.

FIG. 52 illustrates a front view close-up of the aspheric lens and thesubstrate stage of the system of FIG. 51.

FIG. 53 illustrates a software model layout showing the zoom lens andthe aspheric lens.

FIG. 54 illustrates a detail of a software model layout showing the zoomlens and the aspheric lens.

FIG. 55 present zero degree tilt SEM images of cellulose acetatereplicas of nanoholes made at the surface of fused silica by asphericlens machining

FIG. 56 is an irradiance profile of a femtosecond laser pulse with +2 μmLSA (best collimation) as a function of axial position near the focalregion (focal position=1.76 mm).

FIG. 57 is and irradiance profile of a femtosecond laser pulse with +27μm LSA as a function of axial position near the focal region (focalposition=1.711 mm).

FIG. 58 is and irradiance profile of a femtosecond laser pulse with +49μm LSA as a function of axial position near the focal region (focalposition=1.711 mm).

FIG. 59 example methods for casting a nanostructure in accordance withembodiments of the present technology are illustrated.

FIG. 60 example methods for casting a nanostructure in accordance withembodiments of the present technology are illustrated.

FIG. 61 example methods for casting a nanostructure in accordance withembodiments of the present technology are illustrated.

FIG. 62 example methods for casting a nanostructure in accordance withembodiments of the present technology are illustrated.

DETAILED DESCRIPTION

None of the nanofabrication techniques identified herein has so far beenreported to produce large area patterns of high aspect ratio (at least10:1) nanoholes of depths more than 10 μm at high production rates otherthan single-pulse amplified femtosecond laser machining Substrates withuser-defined patterns of high aspect ratio nanoholes of depths more than10 μm can be used in template synthesis of micro/nano structures byreplication. Single-pulse amplified femtosecond laser machining is amethod by which >25:1 aspect ratio and >10 μm deep nanoholes may beproduced. Templates produced by this technique can be used to fabricatestructures by imprinting methods. Also discussed in brief are someapplications where these imprinted structures can be used.

Many different types of materials like metals, alloys, glass,semiconductors, refractories, composites, and polymers have beensubjected to laser treatment and explored for myriad applications. Someexamples of laser materials modification techniques are laser cutting,drilling, welding and brazing, alloying and cladding, surface coating,surface hardening, surface texturing, milling, machining, semiconductordoping, interference lithography, and solid freeform fabrication. Bothcontinuous and pulsed modes of laser operation are used to accomplishthese materials modification processes. In particular, pulsed mode isemployed where high peak intensities are required for materialmodification. Embodiments of the present technology involve ablation ofoptically transparent materials that require high peak intensities forpermanent structural modification.

Femtosecond lasers have been widely used for micromachining of a varietyof transparent materials due to their versatility in producinggeometrically complex structures in three-dimensions with very highprecision. Nonlinear absorption of laser energy by means of multiphotonionization or tunneling ionization followed by avalanche ionizationcauses optical breakdown in transparent materials. Micromachining ofwide band gap transparent materials with femtosecond laser pulses hasbeen reported for applications like binary storage data, opticalwaveguides, waveguide splitters, waveguide optical amplifiers,nanogratings, microfluidics, and flexures. Transparent materials likefused silica, borosilicate glass, soda lime glass, BK7 optical glass,plastic, single-crystal quartz, diamond and sapphire have beensuccessfully micromachined with femtosecond laser pulses.

Fused silica is one of the key transparent substrates used extensivelyfor lab-on-a-chip device applications. Fused silica offers a combinationof properties like wide range of spectral transparency, lowautofluorescence, good biocompatibility, chemical inertness, near zerothermal expansion, excellent thermal shock resistance, and lowdielectric constant and losses. Laser induced breakdown in fused silicawith femtosecond pulses was first reported in 1994. Micromachining offused silica for lab-on-a-chip applications has been accomplished bynumerous investigators.

A highly intense and tightly focused laser pulse from an amplifiedfemtosecond (fs) laser system can induce a permanent structural changein the bulk of a transparent material due to nonlinear absorption.Making use of this nonlinear absorption phenomenon, others havedemonstrated the formation of high aspect-ratio nanoscale diameterholes, or nanoholes, of depths greater than 10 μm at the front surfaceof fused silica by focusing single pulses of temporal length 160 fsusing a high numerical aperture (NA=0.85) microscope objective lens.Hole depths were determined by measuring the lengths of the nanowiresproduced by cellulose acetate (CA) polymer replication (also termed asnanoimprinting) of the patterned fused silica templates, and thenvalidated those measurements by focused ion beam (DualBeam™ SEM/FIB)sectioning of the nanoholes. It was found that the nanoholes' depthmeasurement by CA polymer nanoimprinting slightly underestimates theiractual depth, which makes cellulose acetate polymer nanoimprinting avalid method for nanohole depth estimation. FIG. 1 shows four (4) SEMimages 110, 120, 130, and 140 of nanoholes 102 machined into a fusedsilica substrate using SP-AFL machining FIG. 2 shows two (2) views 210,220 of an array of CA nanowires 230 obtained by nanoimprinting. An SEMimage 300 of the sectioned nanoholes 302 is shown in FIG. 3.

Several authors have reported the formation of nanoholes in transparentmaterials by single-pulse femtosecond laser machining One authorproduced 8 μm deep holes by focusing single shots of temporal length 600fs at the rear surface of a Corning 0211 glass cover slip using amicroscope objective with numerical aperture NA=0.65. Another authorreported the formation of 20-30 μm deep nanoholes at the front surfaceof a Corning 0211 glass cover slip patterned using single-shotfemtosecond diffraction-free Bessel beams. The mechanism(s) responsiblefor the formation of such deep holes by tightly focused single-pulsesfrom an amplified femtosecond laser are still unclear and open todiscussion. A third group proposed the longitudinal spherical aberrationin the focal region of the laser beam caused by the microscope objectivelens combined with self-focusing to be responsible for the formation ofnanoholes, whereas others proposed the role of self-focusing.Furthermore, studies have hypothesized that the laser beam undergoesreshaping upon interaction with the glass surface, which leads to theformation of deep nanoholes. As yet, none of the reported mechanismshave fully expounded the formation of nanoholes.

As stated above, there is no nanolithography technique known yet thatcan produce high aspect ratio (>10:1) and deep (>10 μm) nanoholeseconomically, conveniently, and at high production rate. Although in itsinfancy, single-pulse amplified femtosecond laser machining by White etal. appears to have filled this gap by which >25:1 aspect ratio and >10μm deep nanoholes have been successfully produced at the surface offused silica. Moreover, cellulose acetate replication of these nanoholesunveils a novel-yet-simple imprinting strategy for creating high aspectratio and tall polymer structures. In addition, it is possible to modifythe physical and chemical properties of these imprinted structures byfunctionalizing them to create novel structures. For example, silicananowires can be created by coating a thin layer of silica on top of CAnanowires. Similarly, alumina and titania nanowires can also be created.As these polymer structures have high surface area, are physicallyarranged in a controlled manner, and can be subsequently functionalized,they are useful for a number of applications, such as photovoltaics,high surface area electrodes, and synthetic cell culture substrates.

Nanostructures are routinely fashioned via replication (also termedimprinting) of patterned substrates. Some of the well-establishedreplication strategies that use nanofabricated templates as molds arediscussed earlier herein. Replication strategies used in softlithography and nanoimprint lithography are of particular interest asthey make use of templates with customizable nanoholes, just like SP-AFLmachined templates. CA film replication of SP-AFL machined fused silicasubstrate resembles solvent-assisted micromolding (SAMIM) of softlithography. The only difference being the type of mold used; SAMIM usessoft templates (PDMS molds), whereas White et al. used rigid templates(fused silica molds). Soft molds like PDMS cannot be used with manysolvents because of their affinity towards chemical reactions, such asswelling, which severely limits their application as templates.Nanoimprint lithography uses rigid templates like silicon and fusedsilica, but the replication material reported so far has always been inliquid phase, such as photoresist and PMMA, which is subjected to eitherUV curing or thermal treatment to form the replica. Moreover, theultimate objective of the polymer replica in nanoimprint lithography isto act as a mask to transfer the pattern into the substrate by reactiveion etching. Since nanoimprint lithography is dependent on polymer type,its application as a replication strategy for imprinting polymerstructures is also limited.

This disclosure discusses the capabilities of SP-AFL machined fusedsilica substrates as templates for imprinting polymer structures by filmbased and, in particular, solution based replication strategies. Indeed,solution based replication offers several advantages over film-basedreplication (explained later), which makes it more versatile in itsapplicability. One of the applications of polymer structures imprintedfrom SP-AFL machined templates is in cell biology, where they can beused as synthetic cell culture substrates with tunable topography.

The regulation of cytoskeletal configuration of a cell in response tovariations in extracellular neighborhood is a long establishedphenomenon, and has remained a key research area in developmentalbiology, biomedical engineering, and pharmacology. This regulation isgoverned by sensory mechanisms of the cell that respond to physical andchemical signals arising from the micro/nano environment. The cell canassay these signals and undergo a change in its morphology, conduct,and/or motility; collectively termed morphogenesis. The cellularresponse to externally applied mechanical forces is currently beingexplored by many researchers to understand the physical behavior ofdifferent cell types. Topography, compliance, texture, and anisotropy ofthe extracellular matrix (ECM) or the cell culture substratum affectthese physical forces and influence cell phenotype and activityresponsible for embryogenesis, cell regeneration, and wound healing.Hence, it is essential to understand the characteristics of the physicalinteraction between the cells and their interaction with substratefeatures to further advance cell biology and regenerative medicine.

The effect of substratum topography on cell physiology was predicted asearly as the 1890's, and experimental observations of these effectscoincide with the genesis of cell culture in the 1910's by Harrison.Since then studies on cellular response to solid substrata, alsoreferred to as stereotropism or thigmotaxis, have been actively pursued.Indeed, topographical control of cell behavior has been the subject ofnumerous review articles over the years, focusing on the physicalcharacteristics of the interaction between different cell types anddifferent topographical features. Early investigations on stereotropismwere done on fibrous, mechanically roughened, and mechanically patterned(grooves and ridges) substrata with unrefined definitions. Moreprecisely patterned substrata with good control on topography andgeometric profiles were made available with the advent ofphotolithography and electron beam lithography. Present studies ontopographical control of cell physiology are mostly done by culturingcells on flat substrata with 2D topography in which feature size isnormally in the range 10 nm to 3 μm. 2D topography is obtained bycreating arrays of features like nanogratings, nanopillars, andnanoholes on cell culture substrata via various micro/nanolithographyand nanofabrication techniques. The size scale of these topographicalfeatures is typically smaller than the cell in both lateral and verticaldimensions, and has been found to affect the morphology, survival,adhesion, proliferation, and locomotion of certain cell types. Whenfeatures are considerably larger in vertical dimension and are spaced atleast one cell dimension apart, they tend to behave as physical barriersand provide 3D environments that specifically influence cell morphology,spreading, and alignment by providing more cell-matrix interactionsites, particularly observed in case of fibroblast attachment andmotility.

Cell motility is governed by the polarization of the protrusions(extension and contraction) of its plasma membrane caused bypolymerization and depolymerization of actin filaments. Surfacereceptors like integrins and membrane proteoglycans in the protrusionsfacilitate physical interaction of the cell with its ECM, and provideadhesion to the neighboring stroma. However, presence of any feature orobstacle near the cell is sensed by these receptors, which influencesits protrusions and eventually affects cell attachment, orientation,viability, and motility. The phenomenon of contact guidance is anexample of such a cellular response to topographical features. Frommaterials standpoint, a variety of metals, ceramics, and polymersexhibiting suitable biocompatible properties are used to make substratafor in vitro cell culture studies. Currently, polymers are beingexplored as cell culture substrata as they can be easily and efficientlypatterned via soft lithography with nano/microscale features, forexample polydimethylsiloxane (PDMS). Micropillar arrays of PDMS andsilicon prepared via nanolithography are commonly used to study behaviorof different cell types in 3D micro environments.

Polymer structures imprinted from SP-AFL machined fused silica are agreat candidate as synthetic cell culture substrates to study cellbehavior as they are created by a simple replication technique with veryhigh throughput and reproducibility. In addition, fused silica ischemically inert to organic solvents, so templates made on fused silicalast for a very long time. With solution based replication strategy,structures from virtually any polymer or polymer blend (that makescontinuous films) can be easily imprinted for cell study e.g.,polycaprolactone (PCL). Imprinted polymer structures can be subsequentlyfunctionalized to create bioactive structures, such as silica nanocones,and used in biological applications.

Silica and silica-coated materials constitute a special class ofmaterials, which is used in a variety of biological and biomedicalapplications. Silica is chemically inert to most organic substances,which makes it a suitable host material for many biologicalenvironments. Silica is a well-known catalytic support. In addition,silica can be easily functionalized as the hydroxyl groups (—OH) presentat its surface can covalently bond with other biochemical molecules,such as amino (—NH₂) and carboxyl (—COOH) groups. Functionalized silicaallows further conjugation with other functional groups and biologicalagents, thus making silica a material of choice for manybioapplications. Therefore, materials intended for use in biologicalapplications (e.g., nanoparticles and quantum dots) are often coatedwith silica to improve their biocompatibility. Biosensing, bioimaging,and drug targeting are some examples where functionalized silica isconsistently used.

Nanoscale surface coatings or nanoencapsulation based on silica has alsobeen a subject of great interest. Nanoscale silica has been successfullyproduced by methods like chemical vapor deposition (CVD),catalyst-controlled room temperature chemical vapor deposition,microemulsion, atomic layer deposition, sol-gel synthesis (Stober'smethod), and liquid phase deposition. Silica coatings forbioapplications are most commonly produced by solution-based methods ofsol-gel and microemulsion. However, these methods require prolongedsoaking in chemical solutions, which is unsuitable to imprinted polymerstructures (such as the ones replicated from SP-AFL machined templates)as they can swell or disintegrate. Furthermore, imprinted polymerstructures cannot be exposed to high temperatures as well because theycan oxidize or may even burn. Thus, the only way to coat polymerstructures with silica is by low temperature CVD process. Indeed,coating these polymer structures with any material should be done at lowtemperature to maintain their structural integrity. Besides lowtemperature CVD, imprinted polymer structures can also be functionalizedby sputtering and thermal evaporation methods. Coating processes such assputtering emit fast-moving ions and thermal evaporation emit radiationthat can distort the shape of thin polymer structures, thus cooling thepolymer below its glass transition becomes imperative to sustain theimpact of ions and heat.

Single-pulse amplified femtosecond laser machining has emerged as adirect-write nanolithography technique for patterning transparentmaterials with user-defined arrangement of nanoholes. It is based on theprinciple of nonlinear absorption of photons leading to opticalbreakdown in the bulk of the transparent material. Nanoholes areproduced by tightly focusing amplified femtosecond laser single pulsesthrough a high numerical aperture microscope objective lens at thesurface of the transparent substrate such as fused silica. Celluloseacetate replication and DualBeam™ SEM/FIB cross sectioning of thenanoholes made on fused silica reveal that the holes are >10 μm deepwith aspect ratio >25:1 (White et al.).

CA film replication of the nanoholes produced by this technique by Whiteet al. illustrates a facile method of creating high aspect ratio polymerstructures that are suitable for many applications. Besides polymerfilms, polymer solutions can also be used for replication, which willoffer complete freedom on the final chemistry of the polymer structuresand will also allow replication from thermosets. Replicated polymerstructures can be subsequently functionalized by coating with variousmaterials such as oxide dielectrics and metals. Silica coated polymerstructures can be used in biological applications.

The reason behind the formation of high aspect ratio nanoholes bytightly focused single femtosecond laser pulses is still not clear andopen to discussion. A clear understanding of nanohole formation isimportant to the fabrication of templates as it is to the currentdebate, as it could lead to improved hole parameters, such as diameter,depth, and conical profile. Of all the proposed mechanisms, the role ofspherical aberration on nanohole formation can be investigated byoptical modeling and ablation experiments using an aspheric lens. Anaspheric lens is an optical element with non-spherical surface geometrydesigned to eliminate spherical aberration in the focal region of aperfectly collimated laser beam fully filling the entrance pupil of thelens. Herein, an aspheric lens is used for laser ablation experiments incollimated (no spherical aberration) and non-collimated (with sphericalaberration) conditions to study the role of spherical aberration onnanohole formation.

Fused silica templates prepared by single-pulse amplified femtosecondlaser machining can be used as molds to create polymer structures byreplication. A new replication procedure based on polymer solutions,termed solution casting, is investigated alongside the already knownfilm based replication. Different types of polymer structures areimprinted from these templates to demonstrate the versatility of thereplication procedures.

Modifying the properties of these polymer structures by functionalizingor coating with other materials will further advance their practicalapplications. A low-temperature chemical vapor deposition process isdeveloped to coat the polymer structures with silica to create silicastructures. As silica is used extensively in bioapplications, silicastructures produced by this technique are investigated for applicationsin tissue engineering as cell culture substrates to study cell behavior.

Investigation of the current technology was conducted in part using afemtosecond laser machining system at controlled ambient temperature(21° C.) and atmospheric pressure in a class 1000 cleanroom facility.Mounted on a vibration isolated optical table, the amplified femtosecondlaser machining system includes an amplified femtosecond laser set-up;and a machining workstation, details of which are described in thefollowing paragraphs.

FIG. 4 shows a schematic of the femtosecond laser set-up 400. Theamplified femtosecond laser set-up includes a diode pumped frequencydoubled neodymium-doped yttrium orthovandate (Nd: YVO₄) laser operatingat 532 nm (Coherent, Inc., Verdi V18) 410, a titanium-sapphire laseroscillator (Spectra Physics Inc., Tsunami Model-3941) operating at 76MHz 420, and a 250 kHz titanium-sapphire regenerative amplifier(Coherent, Inc., RegA 9000) 430. The titanium-sapphire laser oscillator420 seeds the titanium-sapphire regenerative amplifier to generateamplified femtosecond laser pulses at the rate of 250 kHz, while boththe oscillator 420 and the regenerative amplifier 430 are pumped by the532 nm green diode laser 410.

The technical specifications of the 532 nm diode-pumped solid statelaser, the titanium-sapphire laser oscillator, and the titanium-sapphireregenerative amplifier are shown in Table 1, Table 2, and Table 3,respectively. The central wavelength of the amplified laser pulse is˜780 nm with a temporal length of 160 femtoseconds. The average power(P_(avg)) of the amplified laser pulses at 250 KHz was measured to be˜1.3 W, which gives the maximum pulse energy (E_(p)) to be ˜5.2 μJ(E_(p)=P_(avg)/250×10³). FIG. 5 and FIG. 6 illustrate the path 510 offemtosecond laser pulses generated by the amplified femtosecond laserset-up 400 travelling through a neutral density filter 520 and a beamexpander consisting of two lenses 530 and 540, and delivered to themachining workstation via a periscope assembly for the machining. Theperiscope assembly 550 consists of two mirrors at 45°, which elevate andguide the laser beam to enter the machining workstation at the rightorientation. The laser pulse energy required for machining is regulatedby adjusting the neutral density filter 520, whereas the distancebetween the two lenses 530 and 540 in the beam expander is adjusted tocollimate the beam. The electro-optic switch within theTitanium-Sapphire regenerative amplifier 430, also called Pockels cell,is externally triggered during machining to enable the emission ofsingle laser pulses for the experiments.

TABLE 1 Technical specifications of the continuous wave pump laser(Verdi-V18, Coherent, Inc.). Output Power >18 W Wavelength 532 nm Linewidth <5 MHz Beam Diameter 2.25 mm ± 10% Beam Divergence <0.5 mrad M²<1.1 Pointing Stability <2 μrad/° C. Power Stability ±1% Noise <0.03%rms Polarization vertical, >100:1

TABLE 2 General specifications of the laser oscillator (Tsunami Model3941, Spectra Physics). Repetition Rate (nominal) 80 MHz Noise <0.2Stability <5% Spatial Mode TEM₀₀ Beam Diameter (1/e²) <2 mm BeamDivergence, full angle <1 mrad Polarization >500:1 vertical

TABLE 3 System specifications of the regenerative amplifier (RegA 9000,Coherent, Inc.). Repetition Rate (nominal) 250 kHz Pulse Width (FWHM)160 fs Polarization linear, horizontal, 500:1 Energy Stability (% rms)<1 Average Power Drift (% rms) <1

The laser pulses delivered by the amplified femtosecond laser to themachining workstation via the periscope assembly are reflected by an 800nm dichroic filter mirror into the microscope objective lens. Themicroscope objective lens focuses the laser pulses onto the surface of afused silica substrate mounted on a stage assembly for machining. Thestage assembly consists of two independent stages capable of moving thesubstrate in XYZ directions with nanoscale precision controlled with aLabVIEW™ program and Aerotech basic software. The stage assemblyconsists of two stages mounted on top of each other: 1) ANT95-3-V forvertical translation (z-stage), and 2) ANT95-50-XY nMT for lateraltranslation (xy-stage). Both the stages are by Aerotech, Inc. and have aresolution of 1 nm. A schematic of the machining workstation is shown inFIG. 7. Large vertical motions (Z direction) are controlled manually andby an additional stepper motor. multimode fiber 701, fiber opticendplate 702, focusing lens 703, long wave pass Raman edge filter 704,12 bit digital camera 705, 9 mm computer TV lens 706, eye piece 707,tube lens 708 and 709, half-mirror 710, white light illuminationproduced from a reflected light vertical illuminator 711, 800 nmdichroic filter mirror 712, 532 nm dichroic filter mirror 713, 532 nmnarrow bandpass filter 714, infinity corrected microscope objective 715,xy-stage 716, z-stage 717, periscope assembly for redirecting andelevating the laser beam 718 and 719.

500 μm thick, double side polished, UV grade fused silica wafers (MarkOptics, Inc.) were used to prepare templates by femtosecond lasermachining Technical specifications of the fused silica wafers used areshown in Table 4. Unless otherwise specified, fused silica substrates ofsize 1 cm×1 cm were diced from large wafers (100 mm diameter) to be usedfor machining. All substrates were cleaned in acetone and isopropylalcohol in order to remove any organic matter and dust from the surfaceprior to machining.

TABLE 4 Technical specifications of the UV grade fused silica used forexperiments. Outside Diameter (O.D.) 100.00 mm ± 0.20 mm Thickness  500μm ± 25 μm Polished Surface Both sides polished Surface Quality (S.Q.)60/40 Surface Roughness 20 Å Total Thickness Variation (T.T.V.) <25 μm

Prior to laser machining, a stylus-based profilometer scans the surfaceof the substrate to obtain a level surface and to map its topography. Itis done in two steps in series. The first step involves the leveling ofthe substrate so that it is nearly normal to the laser beam during lasermachining Leveling is a process of reducing the substrate inclination asa function of stylus extension and compression. It is done by manuallyadjusting the tilt of the substrate in x and y directions while thestylus scans the substrate surface. The tilt adjustment is continueduntil a reasonable leveling or flatness is obtained.

The second step involves fine scanning of the substrate surface toobtain detailed topographical information. The topography arises due tothe presence of hills and valleys (waviness of ˜2 μm) native to thesubstrate surface, which alters its relative position with respect tothe optical focal point, and eventually affects the machined nanoholesize. Thus, it is important that all the points in the patterning areaof interest maintain the same relative position in order to produceidentical nanoholes. The topography data obtained by fine scanning arethen fed to the nanostage during laser machining, which helps maintainthe desired relative position for the entire processing area byadjusting the stage movement. A fine scan of the surface topography isshown in FIG. 8.

Early work in femtosecond laser machining was done with a 60× microscopeobjective lens with a numerical aperture of NA=0.85 and an adjustablecorrection collar. The collar was set to compensate for focusing througha 0.17 mm glass coverslip, but the laser was actually focused on the topside of the substrate, so that the focus exhibited a significant amountof spherical aberration. In the present technology, three differentmicroscope objective lenses were used for machining in air: (1) 60×(NA=0.85; Nikon CF Plan Achromat 79173), (2) 160× (NA=1.25 in water;Leitz Wetzlar Model: ∞/0 PL APO), and (3) aspheric singlet lens(NA=0.68; Thorlabs C330TME-B). Note that the 60× objective lens was usedto focus and machine at the top surface, but the laser beam entering theobjective was collimated, whereas the 160× objective lens was used drywith an input laser beam that was collimated. The 60× and 160×microscope objective lenses were used to make fused silica templates forpolymer imprinting purposes, whereas the aspheric lens was used to studythe role of spherical aberration on nanohole formation.

Patterns are made on the substrate by moving the xy-stage pre-programmedin a LabVIEW™ program. Machining was done by varying two parameters: a)the relative position of the optical focal point with respect to thesubstrate surface (Z); and, b) the pulse energy (E_(p)) of the laserbeam. Optical (visible eye) focus is defined as the focus through theeyepiece on the surface of the fused silica substrate in air withvisible light, and it is different from the focus of the 780 nmwavelength laser. The distance Z is adjusted by moving the z-stage up ordown. The pulse energy (E_(p)) is determined by the formulaE_(p)=P/250×10³, where P is the laser power at 250 kHz measured using apower meter, and is varied by adjusting the variable neutral densityfilter placed in the path of the laser beam, see FIG. 5 and FIG. 6.

Various different types of polymers are used to imprint polymerstructures from single-pulse femtosecond laser machined fused silicatemplates by solution-based replication of the present technology calledsolution casting. The only exception is cellulose acetate (CA)structures, which are usually made by CA films.

A schematic 900 of the CA film imprinting is shown in FIG. 9. CA filmreplication involves wetting the surface of the template 905 withsolvent (acetone 910) followed by placing a piece of 35 μm thick CA film915 on the pattern 920. The solvent softens (or may even dissolve) theCA film 915, and fills the nanoholes of the pattern 920 by the action ofcapillary force. The softened polymer within the nanoholes of thepattern 920 molds against the sidewall of the nanoholes resulting in theformation of a replica 925 of the pattern. Once the solvent dissipates,the imprinted polymer film is gently peeled off from the template 905surface to get the replica, which consists of polymer structures.

FIG. 10 shows a schematic of the solution casting process 1000. Forsolution casting, a polymer solution 1010 prepared by mixing the polymerin a suitable solvent (e.g. polycaprolactone in chloroform) is used. Thepattern 1020 on the template 1030 is covered with a few drops of thepolymer solution and then allowed to dry in normal atmosphericconditions or placed in a vacuum desiccator until the solvent hasdissipated and a film 1040 has formed on the surface. The resulting film1040 is the imprint or the replica 1050 of the pattern 1020, which iscarefully peeled off from the template 1030 similar to CA filmimprinting. Sometimes, surface treatment of the templates 1030 becomesnecessary for the removal (also called lift off) of the polymer imprint1050. Some of the polymer solutions used to make polymer structures byimprinting are shown in Table 5.

TABLE 5 Polymers used to make polymer structures by solution casting,their respective solvents, concentrations, and sources. Polymer SolventWt. % Origin Cellulose acetate (CA) Acetone 2-10 Sigma AldrichPolycaprolactone (PCL) Chloroform 2 Sigma Aldrich PCL-PolyethyleneGlycol Chloroform 2 BME, (PEG) Vanderbilt Polyethylene (PE) Toluene 10 SPI Supplies Polydimethylsiloxane Heptane 25  Dow Corning (PDMS)Polymethylmethacrylate Toluene 5-10 Sigma (PMMA) Aldrich Polyvinylalcohol (PVA) De-ionized 5 Alfa Aesar water Collodion Amyl acetate 2EMS, PA

Polymer structures from the polymers mentioned in Table 5 exceptpolyethylene (PE) can be made by the solution casting process shown inFIG. 10. Polyethylene solution after solvent evaporation does not form afilm, but a loosely connected network of polyethylene powder, which iswhite in color. The template with the white powder network is thenheated to about 155° C. and thermally soaked at this temperature for 2minutes to melt and coalesce to form a transparent and continuouspolyethylene film. The white powder network starts melting around 110°C. and becomes almost clear at a slightly higher temperature. Thetemplate is then cooled slowly after 2 minutes of thermal soaking at155° C. by turning the hot plate off, which takes around 2 hours coolingtime to room temperature before the film is peeled off from thetemplate.

A monolayer of antistick coating based on fluorocarbons is prepared onthe surface of the template prior to imprinting for those polymers thathave a tendency to adhere to the template and affect lift-off of theimprint e.g., PMMA, PDMS, and PVA. In the present technology, antistickcoatings are prepared from 1H,1H,2H,2H-perfluorodecyltrichlorosilane.The template with the hole-side facing upwards is placed in a desiccatorright next to a coverslip glass, and then a drop of fluorosilane isplaced on top of the coverslip glass. As the vacuum is pulled in thedesiccator, the fluorosilane starts to evaporate and coats the templatewith a layer of fluorosilane. A coating similar to Teflon in chemicalstructure, (—CF₂—CF₂—)_(n), is formed.

The dimensions of the imprinted structures depend on the dimensions ofthe nanoholes (lateral and vertical) whereas the dimensions of thenanoholes depend on the machining parameters used during templatefabrication. The depth and the cross-section of the nanohole can bereasonably decreased by reducing the pulse energy. However, in order toincrease the dimensions of the nanoholes, the glass template needs to bechemically etched. Hydrofluoric acid (HF) is known to etch fused silicaisotropically and concentrated aqueous solution of potassium hydroxide(KOH) is known to preferentially attack femtosecond laser affectedregion in fused silica. A combination of isotropic etching in HF andanisotropic etching in KOH can thus be used to enlarge the nanoholes. Inthe present case, the nanoholes were enlarged by sequential etching,first in HF (Lodyne™, BOE, 6:1) and then in 10M KOH at 80° C. Theetching duration depends on the desired dimensions of the etched holes.Etched holes with up to 2.5 μm diameter can be easily produced. Bothas-machined and chemically etched templates are used to imprint polymerstructures in the present study. To distinguish between the two, polymerstructures imprinted from as-machined templates are termednanostructures (e.g., nanowires and nanocones), whereas those imprintedfrom etched templates are termed microstructures (e.g., micropillars).The term nanoimprinting used frequently in this disclosure refers toimprinting of nanostructures.

Imprinted polymer structures such as nanowires can be functionalized bycoating with different materials, mainly noble metals like gold,palladium, and platinum, and dielectric materials like silica, titania,and alumina.

Gold, platinum, and palladium can be deposited by sputtering atcryogenic temperatures (normally when the probe temperature near thesample reaches <−165° C.) in a conventional sputtering machine modifiedwith a cryo-stage connected to a liquid nitrogen container. Thethickness of the metal coating is a function of sputtering time andcurrent applied for the given metal. Usually, 15-20 mA current isapplied for these metals.

Silica can be deposited on CA nanowires in a low temperature chemicalvapor deposition process at 65° C. FIG. 11 shows a pictorial descriptionof the silica deposition process 1100 that takes place in two separateglass beakers 1110 and 1150. First, a silicon tetrachloride solution (˜5mole %) 1112 is prepared by mixing 0.6 ml SiCl₄ and 9.4 ml CCl₄ in a 25ml scintillation vial 1114 with a magnetic stir bar 1116. Thescintillation vial is immediately capped after putting SiCl₄, CCl₄, andthe magnetic stir bar 1116 to avoid any escape of SiCl₄ by vaporization.Two glass beakers, one 1100 with the scintillation vial (1114) and theother vial 1150 with a small quantity of de-ionized water (1152), arethen placed on a digital stirring hot plate 1190 (VWR International,LLC) maintained at 65° C. The beakers 1110 and 1150 are maintained atthat temperature for a minimum of 30 minutes before the start of theexperiments, which ensures the formation of water vapor in beaker 1150.A fresh silicon tetrachloride solution is prepared for each depositionexperiment.

A CA nanoimprint 1118 with erect nanowires prepared from CA film, asdescribed elsewhere herein, is first affixed to an SEM peg 1120 using aLift-n-Press™ sticky tab with the nanowires facing upwards, and then theSEM peg is mounted on a brass holder 1122. The cap of the scintillationvial in beaker 1110 is opened and immediately the brass holder is placedon the neck of the beaker. The beaker is positioned such that the CAnanowires 1118 on the SEM peg 1120 face downward just above thescintillation vial 1114 and are exposed to silicon tetrachloride vapor1124 for adsorption. The exposure time in beaker 1110 (t_(i)) isrecorded right after the brass holder is placed on the beaker. It isvaried from 1 minute to 6 minutes to study the effect of SiCl₄ exposuretime on silica coating thickness, whereas the exposure time in beaker1150 with deionized water 1152 is kept constant at 5 minutes for all theexperiments. It was found that 5 minutes exposure to water vapor inbeaker 1150 is sufficient to hydrolyze all the silicon tetrachloridemolecules adsorbed on the surface of cellulose acetate in beaker 1110;hence, longer exposure times are not required. Silica coated celluloseacetate nanowires are termed silica nanowires or silica nanoneedlesherein.

Nanoholes, etched holes, imprinted polymer structures (nano and micro),and functionalized polymer structures are individually characterized.

Holes produced at the surface of fused silica (as-machined nanoholes andchemically etched microholes), imprinted polymer structures (fromas-machined and chemically etched fused silica templates), andfunctionalized polymer structures were imaged with various fieldemission scanning electron microscopes, most commonly FE-SEM (JSM 6320,JEOL) and FIB-SEM (Lyra 3, TESCAN). As the length of the imprintedpolymer structure corresponds to the depth of the hole, the length ofthe polymer structure is measured in the SEM image to determine thehole-depth. Unless otherwise stated, all imprints are imaged at 45°stage tilt of the SEM. The length of the polymer structure measured inthe SEM image is that of its projection at 45°, so the true length isdetermined by multiplying the projected length by √{square root over(2)}. However, it is true only for those structures that are standingwithout bending or falling over, and only those structures that werestanding were used for calculations.

Silica coating deposited on cellulose acetate nanowires wascharacterized to understand the physical behavior of the depositionprocess and the chemical composition of the coating. SEM images ofsilica nanoneedles produced at different values of exposure time (t_(i))to silicon tetrachloride (beaker 1110) were taken to determine thedeposition rate of silica. The deposition rate was determined bymeasuring the width of the silica nanowires at a projected length of 6μm. Thirty (30) measurements on separate nanowires were made to obtainsufficient data for statistical analysis. The coating thickness (d_(t)_(i) ) was determined using the following formula where, D_(SiO) ₂ ^(t)^(i) is the width of the silica nanowire for the corresponding soakingtime t_(i) and D_(CA) is the mean width of the uncoated celluloseacetate nanowires measured in the same manner on the control sample.

$d_{t_{i}} = \frac{D_{{SiO}_{2}}^{t_{i}} - D_{CA}}{2}$

The standard deviation in the coating thickness (σ_(d) ^(t) ^(i) ) foreach t_(i) was expressed in terms of the standard deviation in the widthof the silica-coated nanowire (σ_(SiO) ₂ ^(t) ^(i) ) and the standarderror in the mean width of the uncoated cellulose acetate nanowire(σ_(CA) ^(m)) using the equation:

$\sigma_{d}^{t_{i}} = \frac{\sqrt{\left( \sigma_{{SiO}_{2}}^{t_{i}} \right)^{2} + \left( \sigma_{CA}^{m} \right)^{2}}}{2}$

An energy dispersive spectrometer (EDS) spectrum of the tip of a silicananowire was taken with a Thermo EDS System 7 on a JSM-7600F thermalFE-SEM. Although EDS information is not quantitative, it does reveal theelemental composition of the coating.

An absorption spectrum of the silica coating was recorded on anattenuated total reflectance—Fourier transform infrared spectrometer(ATR-FTIR model ALPHA-P by Bruker Optics) in the frequency range4000-375 cm⁻¹. The FTIR discerns the presence of different chemicalbonds present in the coating. A small piece of silica coating depositedon gold-coated glass coverslip (5 minutes in beaker 1110) was scratchedto obtain a sample for recording the absorption spectrum. Severalsamples were tested from different silica coating samples (all made at 5minutes) to ensure reproducibility of the results.

A silica nanowire (made at t_(i)=5 minutes) was subjected to focused ionbeam sectioning to see the coating cross-section using a Hitachi NB5000NanoDUE'T FIB-SEM with a gallium current of 20 μA. The cuts were made atdifferent positions of the nanowire starting near the tip of thenanowire to up to 9 μm projected length from the substrate base. SEMimages were continually captured while the cuts were made.

Silica coated cellulose acetate nanowires, or silica nanoneedles, can beused as cell culture substrates to study cell behavior in 3Dmicroenvironment created by silica nanoneedles. Cell culture experimentswere performed using NIH 3T3 mouse embryo fibroblasts. Fibroblasts from3T3 cell lines can be cultured on silica nanoneedles and silica flatsubstrate (as control for comparison), both of can be prepared bydepositing silica on cellulose acetate under similar conditions.

For cell culture experiments, the pattern made on the template can be a2D gradient in nanohole spacing consisting of four identical quadrants,where each quadrant is made up of one gradient pattern of nanoholes. Thepattern can be made by focusing single laser pulses (E_(p)=˜5.2 μJ)through a 160× (NA=1.25 in water) microscope objective lens in air. Ineach gradient pattern, the spacing between the successive nanoholes canbe increased by 1 μm in both x and y directions starting from 10 μm atthe densest location (edge) up to 50 μm at the sparsest location. Thespacing can be reduced by 1 μm per hole from 50 μm to 10 μm to completethe 2D gradient quadrant. FIG. 12 shows a demonstrative picture (not toscale) of the gradient pattern 1200 made on the template. Here, eachblack dot, e.g., 1210, represents a nanohole made by focusing a singlelaser pulse through a 160× microscope objective lens. The pattern 1200is a 2×2 matrix of four quadrants. Each quadrant is formed by increasingthe spacing between successive nanoholes by 1 μm starting from 10 μm inthe densest location to 50 μm in the sparsest location and thendecreasing from 50 μm to 10 μm at 1 μm decrements. A quadrant is amatrix of 84×84 nanoholes, and a 2×2 matrix of these quadrants forms the2D gradient pattern with 168×168 nanoholes i.e., a total of 28,224nanoholes in the pattern. The template thus prepared can be subjected tochemical etching in buffered oxide etch (Lodyne™, BOE, 6:1) for 30seconds followed by rinsing in DI water, and then neutralized in 10M KOHfor 3 minutes followed by rinsing in DI water (all at room temperature)prior to further use. In a sample prepared in this fashion, the depth ofthe nanoholes was determined to be at least 14 μm by cellulose acetatereplication. The average entrance diameter was 750 nm post-etching. Thistemplate with 2D gradient nanoholes is termed wide gradient templateherein. Other gradient patterns are contemplated to facilitate directiongrowth of the cell culture.

A CA replica imprinted from the wide gradient template and a flat CAfilm (both 35 μm thick) can be glued to two separate 170 μm glasscoverslips using a thin layer of uncured PDMS (10:1 mass ratio, Sylgard®184, Dow Corning Corporation) as an adhesive.Flat-CA-on-uncured-PDMS-on-glass-coverslip andCA-replica-on-uncured-PDMS-on-glass-coverslip can be left at roomtemperature for 24 hours for the uncured PDMS to crosslink, which makestheir further handling easier during silica deposition. After PDMScuring, both the glass coverslips can be placed on aluminum SEM pegsusing sticky tabs (Lift-N-Press™, Structure Probe Inc., West Chester,Pa.) and subjected to silica deposition as described herein and shownschematically in FIG. 13. In both the cases, the exposure time in beaker1110 (i.e., to silicon tetrachloride solution) can be kept 5 minutesfollowed by 5 minutes in beaker 1150 for hydrolysis to form silica. Itresults in the formation of 240±40 nm thick layer of silica. Both silicananoneedles and flat silica substrates can be used as cell culturesubstrates to study NIH 3T3 fibroblasts.

Cell culture experiments were performed using the structure describedabove. NIH 3T3 mouse embryo fibroblasts were maintained in Dulbecco'sModified Eagle's Medium (DMEM, Gibco Cell Culture, Invitrogen, Carlsbad,Calif., USA) supplemented with 10% heat-inactivated fetal bovine serum(FBS, Gibco) with 1% penicillin-streptomycin (P/S, Gibco) on tissueculture polystyrene in a water jacketed incubator at 37° C. and 5% CO₂.For experiments, cells were seeded at a density of 80,000 cells/cm² andcultured either overnight or for three days for attachment/viability orimmunocytochemistry experiments, respectively, on both silicananoneedles and flat silica control substrate. For attachment andviability experiments, cells were treated with 4 μM Calcein AM(Molecular Probes, Invitrogen) in phosphate buffered saline (PBS, Gibco)for 15 minutes at 37° C. For immunocytochemistry, cells were fixed with4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X inPBS for 10 minutes, and blocked with 10% goat serum in PBS. Unlessotherwise stated, all steps were performed at room temperature. Cellswere then incubated with phalloidin (Molecular Probes) for 20 minutes or1:100 rabbit anti-mouse a smooth muscle actin primary antibody (αSMA,Abcam, Cambridge, Mass.) overnight at 4° C., followed by treatment of1:50 FITC-conjugated goat anti-rabbit secondary antibody (Abcam) for 2hours. Cells were imaged using a Nikon Eclipse Ti inverted fluorescencemicroscope (Nikon Instruments, Inc., Melville, N.Y.). Images wereanalyzed with Image J (National Institutes of Health, Bethesda, Md.) andn≧50 cells from n≧5 images were used for quantification. Individualcells, not those in aggregates, were quantified for shape descriptors(i.e., average cell area and average cell perimeter).

Cell morphology was further investigated by SEM analysis after cellfixation. For SEM analysis, cells were fixed on silica nanoneedles andflat silica control with 2.5% glutaraldehyde in PBS for 2 hours at roomtemperature followed by rinsing in PBS (×2) and then ultrapure water(×3). Fixed cells were treated with 1% osmium tetroxide in PBS for 1hour at room temperature followed by rinsing in ultrapure water (×3).Fixed cells were then subjected to dehydration, first in graded ethanolin water (30%, 50%, 70%, 95%, and 3×100%) and then in graded ethanol inHMDS (30%, 50%, 70%, 95%, and 3×100%) followed by supercritical drying(Polaron E3000) before SEM analysis. In the present work, unlessotherwise specified, all fused silica templates were patterned at themaximum pulse energy of 5.2 μJ using the laser system explained earlierherein.

The size of the nanoholes patterned by femtosecond laser machining isaffected by the position of the focal spot within the substrate as italters the location of the optical breakdown. In developing the presenttechnology, the effect of the relative position of the optical focuswith respect to the surface of fused silica substrate on the nanoholesize was investigated. The patterning experiment was performed in airusing a 160× microscope objective lens (NA=1.25) at the maximum pulseenergy of 5.2 μJ after leveling the substrate as described elsewhereherein. The sharpest image of the substrate seen through the microscopeobjective lens with the visible light was marked as the optical focus,and its position was recorded as Z=0, where Z is the position of thevertical axis. It must be noticed that the focus of the 780 nmwavelength is different from the visible light focus, and iscomparatively more difficult to determine. In addition, the position ofthe optical focus recorded as Z=0 may vary from user to user as it isdetermined with the naked eye. The relative position of the opticalfocus was varied by adjusting the vertical movement of the substratemounted on a nanostage (z-stage) with respect to the optical focus atZ=0. Nanoholes were patterned on a 500 μm thick fused silica substratecovering a range of vertical motions above and below the optical focus.Referring to FIG. 14, the z-stage was moved at an increment of 1 μm fromZ=−6 μm 1410, to Z=+2 μm 1430, passing through the optical focus at Z=01420. Here, positive values of Z mean the optical focus is above thesubstrate surface by that amount and is obtained by moving the z-stagedown.

SEM images of nanoholes 1500 thus produced and their correspondingcellulose acetate replicas 1600 are shown in FIG. 15 and FIG. 16,respectively. The laser pulse (red) is focused through a microscopeobjective lens onto the substrate (rectangular box) mounted on thez-stage. The stage is moved up and down to change the position of theoptical focus with respect to the substrate surface. Z=0 corresponds tooptical focus on the substrate surface (b); Z=positive corresponds tooptical focus above the substrate obtained by moving the stage down (a),and Z=negative corresponds to optical focus within the bulk of fusedsilica obtained by moving the stage up (c). The blue dotted linecorresponds to the optical focus Z=0.

It can be seen from the SEM images that the relative position of theoptical focus affects the size of the nanohole. In addition, it can beseen that the entrance of some nanoholes is not fully circular, whichcan be attributed to the spatial profile of the laser pulse used formachining. The spatial profile of the laser beam in the present case waselliptical with near-Gaussian intensity distribution. At Z=−6 μm, theposition of the optical focus is so deep into the substrate that thenanoholes do not fully open and remain embedded in the substrate. Thisis possibly due to the inability of the plasma to escape from such adepth.

The nanohole depth was determined by measuring the length of thecorresponding cellulose acetate imprint in the SEM image. Since thelength of the imprint in the SEM image is that of its projection at 45°,the actual length was determined by multiplying it with √{square rootover (2)} as shown in FIG. 17. The dependence of nanohole size (diameterand depth) on the relative position of optical focus with respect to thesubstrate surface is shown as graphs in FIG. 18 and FIG. 19.

Both as-machined and chemically etched fused silica templates, describedelsewhere herein, can be used for solution casting to create polymerstructures. Polymer structures imprinted from as-machined templates aretermed nano, whereas those from etched templates are termed micro todifferentiate between the two. In addition, depending on the morphology,polymer structures imprinted from as-machined templates are referred toas nanowires or nanocones, whereas those from etched templates arereferred to as micropillars herein. The process of solution casting isso versatile that any polymer that forms continuous film, with orwithout the assistance of external energy like heat or UV light, can beused to imprint structures from these templates. Polymer solutions notonly refer to polymers dissolved in solvent, but also polymers that canbe obtained in liquid form e.g., resins. In the present technology,select polymers with different physicochemical properties, but ofcurrent research interest, can be subjected to solution casting tocreate nano- and micro-structures. The following polymers can be used, acommonly used synthetic fiber—cellulose acetate (CA); a ubiquitouslyused linear chain hydrophobic polymer—polyethylene (PE); two biosorbablepolymers—polycaprolactone (PCL) and PCL-polyethylene glycol (PEG); awater soluble polymer—polyvinyl alcohol (PVA); a widely usedmulti-purpose hard plastic—polymethylmethacrylate (PMMA); a 3Dcross-linking polymer—polydimethylsiloxane (PDMS); a commerciallyavailable nitrocellulose—Collodion; a commonly used structuralplastic—polyvinyl chloride (PVC); and a proton conducting ionomer usedin fuel cells—Nafion 117®. All these polymers structures were imprintedfrom the same as-machined and etched templates.

Polycaprolactone (PCL) is a low melting point biodegradable polymer.Chemically, it is a polyester of molecular formula (C₆H₁₀O₂)_(n) and issynthesized by ring opening polymerization of ε-caprolactone in thepresence of stannous octoate. PCL finds applications in biomedicalengineering such as long term implants and (targeted) drug deliveryvehicles because of its slow degradation in physiological conditions bythe hydrolysis of its ester linkages. PCL is hydrophobic in nature andis often copolymerized with hydrophilic polymers (e.g., polyethyleneglycol and hyperbranched polyglycidol) to synthesize amphiphilic blockcopolymers. In the present technology, nanowires and micropillars of PCLand PCL-8 wt % PEG can be made by solution casting of their respectivesolutions made in chloroform. FIG. 20 and FIG. 21 show SEM images of PCLnanowires and micropillars, respectively, as prepared by the methodsdescribed herein. PCL-PEG nanowires and micropillars are shown in FIG.22 and FIG. 23, respectively.

Chemically, cellulose acetate is the acetate ester of cellulose, anaturally occurring polymer. It is mostly used in the form of films andfibers. Cellulose acetate films were first introduced in photography asa replacement for the highly flammable nitrocellulose films as basematerials for photographic emulsions. Later, cellulose acetate filmswere shown to be very useful as a replication material in microscopy formetallographic studies of metallic and biological samples. Indeed, it isknown to use microscopy grade cellulose acetate films to determine thedepth of femtosecond laser machined high aspect ratio nanoholes byreplication. Cellulose acetate films can replicate these deep featureswithout any observable stretching. Cellulose acetate is also used as asemi-permeable membrane in separation processes and films/filters inbiomedical applications. One of the most common methods for thepreparation of cellulose acetate fibers is electrospinning, and thecellulose acetate fibers thus produced in the form of mats have beenexamined as antimicrobial surfaces and drug delivery agents. The size ofelectrospun cellulose acetate fiber is usually controlled by adjustingthe solvent system, and fibers with diameters in the range 100 nm to 5μm have been successfully fabricated. In the present technology, ACSgrade acetone can be used as the solvent to prepare cellulose acetatesolution for solution casting. It was found that a 5 wt % celluloseacetate (average molecular weight ˜30,000) solution can form films thatcan be peeled off from the template. Dilute solutions of celluloseacetate (e.g., 2 wt %) can also be used to form peelable films bycasting multiple layers. FIG. 24 and FIG. 25 show cellulose acetatestructures imprinted from as-machined templates (CA nanowires) andetched templates (CA micropillars), respectively.

Polyethylene is one of the most widely used plastic materials in theworld with majority of its applications in packaging industry. It isproduced by polymerization of ethylene and its chemical formula isusually (C₂H₄)_(n). It is further classified into several categoriesdepending on the density and degree of branching. The two classes ofpolyethylene used predominantly are high-density polyethylene (HDPE) andlow-density polyethylene (LDPE). HDPE (ρ≧0.941 g/cm³) has strongintermolecular forces and high tensile strength due to a low degree ofbranching, whereas LDPE (ρ=0.91-0.94 g/cm³) has weak intermolecularforces and low tensile strength due to a high degree of short and longchain branching. HDPE is generally used to make structures andcomponents for storage applications, whereas LDPE is commonly used tomake films and rigid containers. Besides HDPE and LDPE, ultra highmolecular weight polyethylene (UHMWPE) is also used in a variety ofapplications because of its high toughness in addition to excellentchemical resistance and low wear rate. UHMWPE also finds applications inbiomedical engineering to make parts for hip and knee implants. Althoughit is always coated with hydroxyapatite or calcium phosphate to improveits cytocompatibility, it was recently shown that nanostructuredpolyethylene shows increased osteoblast and endothelial cell adhesionand can be explored for its applications in orthopedic and vascularapplications. In embodiments of the present technology, nanowires andmicropillars of polyethylene can be imprinted from the templates bysolution casting in which an additional heating step can be used. Theprocess is described elsewhere herein. SEM images of polyethylenenanowires and micropillars are shown in FIG. 26 and FIG. 27,respectively.

PVA is a non-toxic and water-soluble synthetic polymer with molecularformula (C₂H₄O)_(n). The structural monomer of PVA is vinyl alcohol,which is unstable. It only exists in its tautomer form acetaldehyde,which is a more stable compound. Thus, PVA is not prepared bypolymerization of its monomer like other polymers. Instead, it isprepared from polyvinyl acetate (PVAc) by hydrolysis where the esterlinkages in PVAc are substituted with hydroxyl groups. Some common usesof PVA are: preparation of contact lens and polarizer, water transferprinting, and as a packaging material in food industry. PVA is also apotential biomaterial. In embodiments of the present technology, PVA(mol. wt. ˜30,000) structures can be made from its solution prepared inDI water, and are shown in FIG. 28 and FIG. 29.

PMMA is a transparent polymer with molecular formula (C₅O₂H₈)n. It is alightweight thermoplastic with good resistance to shattering, and isoften used as an alternative to inorganic glass and polycarbonate. It isthus also called acrylic glass. PMMA is usually chemically modified forpractical purposes. Due to its low cost and transparent nature, PMMA isused in a variety of applications, such as lenses and windows forautomobiles and aircrafts, viewing ports in underwater vehicles,aquariums, daylight redirection systems, and aesthetic structures.Because of its good compatibility human tissue, PMMA is also used inmedical technologies and preparation of biological implants e.g.,cosmetic and orthopedic surgery. PMMA is also found to be an attractivematerial for microfluidic lab-on-a-chip devices. In embodiments of thepresent technology, PMMA structures can be imprinted using its solutionin toluene. SEM images of PMMA nanowires and micropillars are shown inFIG. 30 and FIG. 31, respectively.

Collodion is essentially a solution of nitrocellulose. It findsapplications as a glue to attach electrodes to the head forelectroencephalography, cleaning agent for telescope optics, and as areplication material in microscopy. In embodiments of the presenttechnology, Collodion structures can be imprinted using its commerciallyavailable 2% solution in amyl acetate (Parlodion). FIG. 32 and FIG. 33show the SEM images of Collodion nanowires and micropillars,respectively.

PDMS is also a transparent polymer and it belongs to the family oforganosilicon compounds called silicones. Its molecular formula is(C₂H₆OSi)n. It is hydrophobic, non-toxic, not flammable, heat resistant,and inert to most chemical substances. It finds applications as contactlenses and medical devices. PDMS is currently a material of choice formicrofluidic lab-on-a-chip devices and elastomer stamps for softlithography. Commercially available two-part silicone elastomer kitSylgard 184 is the most common material used to prepare PDMS. The twoparts, base and curer, are usually mixed in 10:1 mass ratio and curedeither in room temperature or at elevated temperature (usually 80° C.for 2 hours) for cross-linking. The mechanical properties of PDMS can bealtered by varying the base to curer ratio. Uncured PDMS mixture is tooviscous to fill the nanoholes of the templates, so its viscosity can bereduced by mixing in n-heptane. In embodiments of the presenttechnology, PDMS structures can be imprinted using a 33 wt % PDMS (10:1base to curer ratio) solution in n-heptane. SEM images of PDMS nanowires(nanocones) and micropillars are shown in FIG. 34 and FIG. 35,respectively.

PCL and PCL-PEG structures imprinted from as-machined fused silicatemplates are shown in FIG. 20 and FIG. 22, respectively. The imprintedstructures appear to be 30-40 μm long nanowires compared to ˜15 μm deepnanoholes they are imprinted from. Apparently, these nanowires undergo alot of stretching during the imprint lift-off process. It can bereasonably explained with the help of various forces acting on thepolymer within the nanoholes during their lift-off. The adhesivestrength between the glass nanohole and the polymer exceeds the tensilestrength of the respective polymers (PCL and PCL-PEG). Consequently, thepolymers yield during their lift-off, which results in their stretching.PCL and PCL-PEG nanowires stretch even when the templates are coatedwith antistick to help release the imprint. Furthermore, freezing thePCL and PCL-PEG imprints below their glass transition temperature (<−60°C.) before lift-off also does not avoid their stretching. Thesenanowires were measured to be around 100 nm wide near their tip. Sincethe wires come out very long (>30 μm) due to stretching and are notstiff enough to carry their weight, they fall over acquiring theappearance of nanocarpets or nanomats. The stretched and fallen PCL andPCL-PEG nanowires have very high surface area and aspect ratios greaterthan 200:1.

SEM images of CA, PE, PVA, PMMA, and Collodion structures imprinted fromas-machined templates are shown in FIG. 23, FIG. 26, FIG. 28, FIG. 30,and FIG. 32, respectively. The dimensions of these structures are fairlyclose to that of the nanoholes and morphologically they appear asnanowires or nanocones with their base diameter around 750 nm and tipdiameter around 150 nm. It can also be seen that most of the polymernanocones/nanowires slightly bend in one common direction whereas sometend to bend in random orientations. The yield of the imprintedstructures (i.e., the total number of imprinted structures with respectto the total number of nanoholes in the pattern) can be very high asmost of the nanoholes are successfully replicated. However, some polymernanocones tend to break (CA and PE) or stretch (PMMA) during lift-off.The yield, to a certain degree, can improve if adequate time is givenfor the solvent to dissipate before the imprint lift-off. Most PDMSnanocones tend to fall over unlike any other polymer nanocones, butwithout breaking. It can be attributed to the low Young's modulus ofPDMS, which is <1 MPa for any given base to curer ratio. Moreover, thechemical structure and mechanical properties of PDMS are very differentfrom other polymers discussed herein. Its 3D cross-linked structuretogether with a combination of very long Si—O skeletal bond,unobstructed skeletal oxygen atoms, and large Si—O—Si bond angle of 143°provide it with extraordinary dynamic flexibility and deformability. Thepolymer structures imprinted from the etched template adopt the shapeand dimensions very close to that of the cylindrical holes they are castfrom. Morphologically, they appear as micropillars though their texturesvary because of their physicochemical properties. The yield can approach100% in all the polymers.

Thin film imprinting is suitable for those polymers that can be obtainedor prepared in the form of thin films (e.g., cellulose acetate andpolyvinyl chloride) and at the same can be softened by a readilyavailable solvent, thus making it a highly selective process. Thus,polymers that do not soften or dissolve in any solvent cannot be used tomake polymer structures by thin film imprinting e.g., thermosets likepolydimethylsiloxane (PDMS). In comparison, solution casting can be usedto imprint polymer structures from virtually any polymer that formscontinuous films. Blending different polymers or reinforcing polymerswith other polymers or non-polymeric materials leads to the formation ofmultifunctional polymers with novel properties for a variety ofapplications in particular biomedical engineering. Such multifunctionalpolymer structures with varying chemical composition and tunablephysical properties can be more efficiently prepared by solution castingthan thin film imprinting. Furthermore, heterogeneous structurescomposed of multiple layers of different polymers can be prepared bysolution casting, which are otherwise not possible with thin filmimprinting. Thus, solution casting offers several advantages over thinfilm imprinting, and can be employed for fabrication of novel polymerstructures for advanced technologies.

Polymer structures imprinted from templates can be functionalized bycoating with different materials in order to modify their physical,chemical, and biological properties. Functionalized polymer structuresare useful for a number of applications e.g., photovoltaics, highsurface area electrodes, and synthetic cell culture substrates. Inembodiments of the present technology, polymer structures can be coatedwith noble metals (gold, platinum, and palladium) by sputtering, andwith silica by chemical vapor deposition.

Noble metals can be coated at cryogenic temperatures in a sputteringmachine as described elsewhere herein. The thickness of the metalcoating can be varied by changing the sputtering time and appliedcurrent/voltage. However, sputtering is a line-of-sight depositionprocess, which affects the metal coating coverage on the surface of theimprinted polymer structures. The sidewalls of the polymer structurereceive considerably less amount of metal coverage when compared to thetips of the polymer structures. Nevertheless, the coating coverage canbe improved by tilting and spinning the polymer imprints for which thesputtering instrument will need a stage for sample manipulation. All theSEM images of the polymer structures shown herein are taken aftercoating with noble metals, mostly platinum, below −165° C. Besides noblemetals, the imprinted polymer structures can be coated with othermaterials as well, such as insulators and metals, provided thedeposition temperature is low enough to avoid polymer softening ordisintegrating.

In embodiments of the present technology, a low temperature chemicalvapor deposition process can coat cellulose acetate nanowires withsilica to prepare silica nanowires (or nanoneedles). It is a two-stepchemical vapor deposition, which deposits a thin layer of silica atopthin film imprinted CA nanowires at 65° C. by hydrolysis of silicontetrachloride (SiCl₄). Two separate beakers can be used for silicadeposition: one with silicon tetrachloride solution and the other withwater. The surface of CA nanowires can be covered with SiCl₄ in thefirst beaker, which cam be hydrolyzed to form SiO₂ in the second beaker.The silica coating thickness can be studied as a function of exposuretime in beaker 1110. The exposure time in beaker 1150 can be keptconstant at 5 minutes, which is sufficient for the complete hydrolysisof SiCl₄ molecules adsorbed at the surface of CA nanowires in beaker1110. The coating thickness can be determined for the first 6 minuteswith the help of SEM images of the corresponding silica nanowires.

The rate of silica deposition as a function of exposure time in beaker1110 (SiCl₄ solution) is shown in FIG. 36. FIG. 37 shows an SEM image ofan array 3700 of silica nanowires obtained after 5 minutes exposure inbeaker 1110. In order to confirm the formation of silica, a silicananowire can be subjected to elemental analysis using EDS. The EDSanalysis, spectrum 3800 shown in FIG. 38, confirms the presence ofsilicon and oxygen. It reveals the presence of silicon and oxygen in thecoating. The sample was coated with palladium prior to SEM/EDS analysis,thus shows up in the EDS spectrum. Although it is a qualitativemeasurement, the EDS spectrum does reveal the presence of silicon andoxygen in the coating, thus confirming the formation of silica. Tofurther characterize the silica coating, an absorption spectrum can berecorded on an ATR-FTIR and is shown in FIG. 39.

The mid-infrared absorption spectrum of the coating shown in FIG. 39reveals the presence of various vibrational modes in silica, and givesmore information about its chemical structure. It shows the presence ofvarious Si—O—Si and O—H vibration modes. The broad band in the interval3700-3000 cm⁻¹ 3910 corresponds to the intensive overlapping of thestretching modes in hydrogen-bonded hydroxyl bands produced by O—H bondsin water and Si—OH. A small peak at ˜1635 cm⁻¹ 3920 is usuallyattributed to the deformation vibrations of the O—H groups in physicallyadsorbed molecular water at the surface of silica coating and its weakintensity confirms the presence of very low amount of molecular water inthe coating. A sharp peak at ˜927 cm⁻¹ 3930 appears due to thestretching vibrations of Si—OH or Si—O⁻ groups in silica, whichindicates the presence of a large number of silanol groups on thecoating surface. The band at 1000-1300 cm⁻¹ 3940 appears due to theanti-symmetric stretching of Si—O—Si bonds in silica. This band can beresolved into a peak at 1041 cm⁻¹ (TO₁) and a shoulder around 1156 cm⁻¹(TO₂). The TO₁ peak in silica is usually found at 1082 cm⁻¹ and itsshifting towards lower frequencies, 1041 cm⁻¹ in the present case, hasbeen reported to be associated with oxygen deficiency and lowerthree-dimensionality in the structure of silicon oxide. It indicatesthat the stoichiometry of the silica coating in the present case isSiO_(x) with x<2. Furthermore, the peak appearing at 799 cm⁻¹ is due tothe bending vibration of Si—O—Si bonds. A peak due to the bending modesin Si—OH bonds also appeared around ˜790 cm⁻¹ 3950; however, it is veryclose to the Si—O—Si bending mode and cannot be seen clearly in thespectrum. A series of peaks appears in the lower frequency region from448 to 424 cm⁻¹ 3960, which could have arisen due to the combination ofbending and rocking modes of Si—O—Si or O—Si—O bonds in the coating.However, it is very difficult to resolve and identify all these peaks indetail.

A silica nanowire produced by coating for 5 minutes was sectioned usinga focused ion beam instrument to view its cross-section. A series of SEMimages were captured while sectioning the silica nanowire, and are shownas a composite picture in FIG. 40. The tip of the silica nanowire wascut with a gallium current of 20 μA at the projected length of ˜9 μmfrom the base (i.e., ˜13 μm actual length). The inset (A) shows the CAnanowire 4010 at the center of the structure. A sequence of cuts wasmade further down the silica nanowire; two of them are shown in (B) and(C), where the silica coating 4020 and CA 4010 can be easilydistinguished by contrast. In addition, the silica coating thickness andCA nanowire diameter can also be clearly seen as well. The FIB millingangle was slightly changed to drill into the CA nanowire and reveal theinterior of the silica nanoneedle 4020 (D). It indicates that the silicacoating is around 125 nm thick and the CA nanowire is 175 nm in diameterat that particular location on the silica nanowire. It must be noticedthat the FIB-sectioned hole appears elliptical because it was imaged at58° from the horizon in the FIB/SEM.

FIG. 41 and FIG. 42 show the SEM images of the nanoholes, e.g., 4110from an edge of the 2D gradient pattern and silica nanoneedles 4210,respectively. The average diameter of the nanoholes is 750 nm and thedepth ˜14 μm. Flat silica coated cellulose acetate and silicananoneedles can be used as cell culture substrates to cultivate NIH 3T3fibroblasts.

The cellular response can be altered in the presence of silicananoneedles as compared to flat silica surfaces. It can be seen in FIG.43 that the fibroblasts 4310 “ball up” or agglomerate on flat surfaces(A), whereas they extend their foci to adhere to the nanoneedles (B),e.g., 4320. The figure shows how the morphology of NIH 3T3 fibroblastsis affected by the surface topography of the substrate. Cellagglomeration on flat silica is an indication of poor adhesion, whereascell spreading on silica nanoneedles is an indication of good adhesion.Silica coating cracks during cell fixation and can be seen in these SEMimages. As shown, cell adhesion to surfaces with silica nanoneedles canbe an order of magnitude higher than that on to flat surfaces (p=0.005,FIG. 44, while viability can be maintained for cells adhered to eithersurface (FIG. 44(B)). Fibroblast spreading also can be influenced in thepresence of silica nanoneedles. Fibroblasts can exhibit a larger areaand perimeter on silica nanoneedles than those cultured on flat surfaces(p<0.005 and p=0.007, FIG. 44(C) and FIG. 44(D), respectively).

The presence and spacing of silica nanoneedles can influence competitivecell-cell versus cell-matrix interactions and can be seen in FIG. 45.Flat surfaces allow for the formation of large, unorganized cellaggregates and independent, single-cell growth (A-B). Cells interactdirectly with the needles and only small cell aggregates, if any, areable to form (C-D). Parallel rows of tightly spaced needles sequestercells between them (E). Few, if any, cells are able to attach in areasof densely packed needles, but attachment improves as spacing becomessparser (F). Cells either spread out individually or formed large,multicellular aggregates (A) on flat silica substrates that do notdisplay noticeable organization or regularity (B). In comparison, thepresence of silica nanoneedles can prevent cell aggregation, interruptcell-cell contacts, and promote interactions with the matrix (C-F).

Note that the fibroblasts can preferentially align themselves alongtightly spaced nanoneedles in gradient regions with sparse spacing inthe orthogonal direction. In regions with sparse distributions in bothdirections, cells can interact both with adjacent needles as well asthose located in the parallel rows (C-D). In general, parallel rows ofsilica nanoneedles with substantial spacing between them (i.e. >30 μm)can sequester cells and bias them towards cell-matrix interactions. Thisspacing also can control the size and geometry of small cell clusterspresent (E). Threshold spacing between silica nanoneedles appears toexist that can regulate the ability of cells to adhere and spread in twodimensions (F).

Relative spacing ratios for local regions of needles can regulate cellspreading. Sparse spacing in two dimensions can allow single cells tospread in two dimensions, whereas tight spacing in one dimension butwide spacing in the orthogonal direction can influence cells to spreadonly in one direction (see insets in FIG. 4.4( e)C). Cells tend tospread in one dimension when the ratio between nanoneedles spacing inorthogonal directions is less than 0.5 (given that the nanoneedlespacing can allow for cells to attach between parallel rows). Incontrast, the cells tend to spread in two dimensions when thenanoneedles spacing ratio is greater than 0.5. No significant differencein αSMA expression between flat silica and silica nanoneedles substrateshas been observed. Furthermore, αSMA stress fibers are not observed oneither substrate.

In embodiments of the present technology, silica nanoneedles can act asa means to modulate cell-cell versus cell-matrix interactions.Competitive cell-cell and cell-matrix interaction is a fundamentalregulator of embryogenesis and has been manipulated for complex tissueengineering strategies. Cells of different lineages exhibit biasedinteractions depending upon their physiological location and function.For example, sheet-like epithelial and endothelial cells are dominatedby cell-cell contacts through which various signals can be propagated;yet, cells of a mesenchymal lineage are primarily cell-matrixinteractive. Intricate lineage transition events, such asmesenchymal-to-epithelial transition (MET) and its reverse (EMT), can beinfluenced by shifting the balance between these competitive events. Incertain cancers, malignant cells in a primary tumor can undergo such atransition, allowing them to escape from their current location andmetastasize. By probing and modulating cell adhesion events, fundamentalprocesses involved in embryogenesis, tissue engineering, and canceroustransformation can be intimately studied, controlled, and exploited fortherapeutic benefits.

In embodiments of the present technology, the presence of silicananoneedles can improve cell adhesion to the surface and stimulate cellspreading without substantially affecting viability. Qualitativeanalysis comparing cellular response to flat silica and silicananoneedles is shown in FIG. 44. Silica nanoneedles were found topromote cell attachment and spreading. Significantly more cells attachto nanoneedle-containing surfaces (A) while cell viability is unaffected(B). Cell spreading (area, perimeter) are increased onnanoneedle-containing surfaces (C and D). It can be seen that directinteractions between fibroblasts and the silica nanoneedles caninterrupt the formation of large cell aggregates that can be seen onflat silica surfaces. Parallel rows of nanoneedles effectively canconstrain the cells and regulated the geometry of small aggregates thatform (FIG. 45). Furthermore, the cellular response can be dependent uponnanoneedles spacing. The ratio of nanoneedles spacing in orthogonal rowscorrelates with cell spreading in one versus two dimensions, suggestingthat the cells may actively sense the 3D microenvironment and thatnanoneedles spacing can be used as a parameter to regulate cellbehavior.

Referring to FIG. 59 through FIG. 62, methods of the present technologyare illustrated. Referring to FIG. 59, a method 5900 for solutioncasting a nanostructure is illustrated. In such method, a template canbe prepared by ablating nanoholes in a substrate usingsingle-femtosecond laser machining—Block 5910. The nanoholes can bereplicated by applying a solution of a polymer and a solvent into thetemplate—Block 5930. After the solvent has substantially dissipated(Block 5940), the replica can be removed from the substrate—Block 5950.In some embodiments, the polymer solution comprises one of: celluloseacetate in acetone, polycaprolactone (PCL) in chloroform,PCL-polyethylene glycol in chloroform, polydimethylsiloxane in heptane,polymethylmethacrylate in toluene, polyvinyl alcohol in de-ionizedwater, and collodion in amyl acetate. In some embodiments, the polymeris capable of forming a continuous film. In some such continuous filmembodiments, the polymer is capable of forming a continuous film throughthe application of external energy. In some embodiments, the polymersolution comprises a two percent (2%) solution by weight of one of:cellulose acetate in acetone, polycaprolactone (PCL) in chloroform,PCL-polyethylene glycol in chloroform, and collodion in amyl acetate;while in other embodiments, the polymer solution comprises a solutionbetween two percent (2%) and ten percent (10%) by weight of celluloseacetate in acetone. In yet further embodiments, the polymer solutioncomprises a twenty five percent (25%) solution by weight ofpolydimethylsiloxane in heptane; or a solution of between five percent(5%) and ten percent (10%) by weight of polymethylmethacrylate intoluene; or a five percent (5%) solution by weight of polyvinyl alcoholin de-ionized water.

Referring to FIG. 60, and continuing to refer to FIG. 59 for context,further example methods 6000 for casting a nanostructure in accordancewith embodiments of the present technology are illustrated in someembodiments, prior to applying a solution of a polymer and a solventinto the template (Block 5930), a fluorocarbon-based antistick coatingcan be applied to the template—Block 6020. In some such embodiments, thefluorocarbon-based antistick coating is prepared fromperfluorodecyltrichlorosilane.

Referring to FIG. 61, an example method 6100 for casting a nanostructureis shown. In such methods, a template can be prepared by ablatingnanoholes in a substrate using single-femtosecond laser machining—Block6110. The nanoholes can be replicated by applying a polymer resin intothe template—Block 6130. After allowing the resin to set (Block 6140),the replica can be removed from the substrate—Block 6150.

Referring to FIG. 62, an example method 6200, for casting ananostructure is shown. In such methods, a template can be prepared byablating nanoholes in a substrate using single-femtosecond lasermachining—Block 6210. The nanoholes can be replicated by casting asolution of a polyethylene and a solvent into the template—Block 6230.After the solvent has been allowed to substantially dissipate (Block6240), the polyethylene as cast in the template can be melted throughapplication of energy—Block 6250. After allowing the melted polyethyleneto cool (Block 6260), the cooled polyethylene replica can be removedfrom the substrate. In some such embodiments, the polymer solutioncomprises ten percent (10%) solution by weight of polyethylene intoluene. In some such embodiments, melting comprises heating to aboutone hundred fifty five (155) degrees Celsius for about two (2) minutes;and cooling comprises cooling at room temperature for at least about two(2) hours.

The nanostructures produced by the methods illustrated in FIG. 59through FIG. 62 can find application in a wide variety of fields asdisclosed herein throughout.

Consider two issues with regard to single pulse femtosecond lasermachining First, the depth of the nanoholes produced can be greater thanthe depth of focus of the laser beam, i.e., the depth over which thebeam remains tightly focused (measured as the depth over which the areaof the beam increases by less than a factor of two). Second, the plasmaformed due to optical breakdown and avalanche ionization, under mostconditions, can become over-dense, opaque, reflective, andself-limiting, resulting in the formation of a shallow crater. Thus,mechanisms are needed to explain the formation of high aspect ratio anddeep nanoholes. Three mechanisms have been proposed by differentresearch groups: spherical aberration, self-focusing, and beamreshaping. These proposed mechanisms are not exclusive and may all playa role to some degree in the formation of deep, high aspect ratiofeatures.

Spherical aberration is an optical phenomenon in which light rayspassing through a spherical lens focus at different locations along theoptical axis to cause to a prolonged focal region. The effect ofspherical aberration on the formation of high aspect ratio nanoholes canbe reasonably investigated using an aspheric lens. An aspheric lens hasa non-spherical surface geometry designed to greatly reduce sphericalaberration in the focal region of a perfectly collimated laser beamfully filling its entrance pupil. Thus, an aspheric lens (NA=0.68;Thorlabs, C330TME-B) was used for machining experiments and opticalmodeling. This particular aspheric lens was chosen because of its highnumerical aperture and the availability of its detailed specificationsand optical coefficients required for optical modeling.

Aspheric lens machining of fused silica can be performed with themachining platform presented earlier. The lenses of the beam expander,shown in FIG. 6, can be adjusted so that the laser beam fully fills theentrance pupil of the aspheric lens. The entrance pupil of the asphericlens is 5 mm in diameter. The machining can be performed at differentrelative positions of the optical focus with respect to the substratesurface (Z) as explained elsewhere herein. The laser pulse energy can bekept constant at 5.2 μJ. SEM images of nanoholes made on a fused silicasubstrate by aspheric lens machining and their corresponding celluloseacetate (CA) replicas are shown in FIG. 46 and FIG. 47, respectively. Itcan be seen from these replicas that the depth of nanoholes decreaseswhen the laser beam fully fills the entrance pupil of the aspheric lens.The deepest nanoholes can be around 4 μm for Z=+52 to Z=+60 from theirCA replicas. The cellulose acetate replicas show that the aspheric lensmachined nanoholes are not as deep as those machined by the 60× and 160×microscope objectives. The aspheric lens can minimize the sphericalaberration in the focal region of the laser beam even when its entrancepupil is fully filled. Thus, the formation of shallow nanoholes (maximum4 μm) by aspheric lens machining might be expected to be due to thereduced spherical aberration in the focal region of the laser pulse.However, it can be seen from the SEM images in FIG. 46 that the singleamplified femtosecond laser pulse undergoes multiple fragmentations uponfocusing through the aspheric lens to form several nanoholes arranged ina crescent shape that resembles the structure of a cat's paw (from Z=+52to Z=+60). The formation of crescent-shaped laser damaged regions may bebecause the aspheric lens has a high numerical aperture and hence isvery sensitive to minor beam misalignments, which may result in opticalaberrations in the focal region. In fact, it is known that misalignmentof a high-NA lens results in comatic aberration or coma.

Three configurations were modeled in an optical design softwareapplication (Zemax by Radiant Zemax, LLC) to demonstrate that the causeof the structures is misalignment of the aspheric lens. Theseconfigurations are based on the propagation path of the laser beam(i.e., laser beam axis) with respect to the optical axis of the asphericlens and are schematically shown in FIG. 48. The configurations modeledwere: ideal condition (i.e., the laser beam axis coincides with theoptical axis), the laser beam decentered 0.5 mm in both X-axis andY-axis, and the laser beam tilted at an angle of 0.5° to the opticalaxis. In the model A) ideal condition: the laser beam axis is parallelto the optical axis of the aspheric lens and travels through the centerof its entrance pupil, B) the laser beam axis is decentered 0.5 mm eachin X and Y directions, and C) the laser beam axis is tilted by 0.5° tothe optical axis of the lens. The red line shows the axis of the laserbeam, where the actual laser pulse fully fills the entrance pupil of theaspheric lens.

The laser beam intensity profiles at and near the focal region for theaforementioned conditions were estimated by Zemax modeling. Twoassumptions were made: the spatial profile of the laser beam isperfectly Gaussian; and, the entrance pupil of the aspheric lens isfully filled with the laser beam (i.e., the 1/e² intensity radius of theGaussian beam equals the 2.5 mm pupil radius of the lens).

FIG. 49 and FIG. 50 show the focal spot diagrams and the correspondingHuygens point spread functions (PSF), respectively, for the threeconfigurations modeled in Zemax. The PSF was obtained using a fastFourier transform (FFT) function built-in in the Zemax. A represents theideal condition; B represents a 0.5 mm decenter each in X-Y, and Crepresents 0.5 degree tilt with respect to the optical axis of the lens.The black circle represents the Airy Disk. The scale bar and RMS are inmicrometers (μm). The spot radius is within the diffraction limit in theideal condition, as indicated by the rays falling within the Airy disk(black circle) in FIG. 49 (A), and its root mean square value (RMS) of˜0.21 μm being less than the radius of the Airy disk. Similarly, thespot radius in the second case (the laser beam is decentered 0.5 mm eachin X and Y directions) also appears to be within the diffraction limit,but with a slight increase in its RMS value to ˜0.27 μm. The shape ofthe spot, however, is no longer completely circular and shiftsmarginally in the XY directions; some rays form elliptical shapes butyet remain within the Airy Disk as seen in FIG. 49 (B). It indicatesthat decentering the laser beam up to 0.5 mm each in X and Y directionsis still close to the ideal condition. FIG. 49 (C) shows the spotdiagram when the laser beam arrives at an incidence angle of 0.5° withrespect to the optical axis of the aspheric lens. It can be seen thatthis slight tilt can result in pronounced comatic aberration as the spotradius is larger than the diffraction limit, and the spot radius RMSvalue has increased to ˜6.5 μm (nearly 30 times larger than that in theideal condition). This comatic aberration (or coma) due to tilting ismost likely the cause for the laser damage region to acquire thecrescent shape, see FIG. 46.

FIG. 50 shows the surface contours of the Huygens point spread functions(PSF) for the three configurations. The scale bar is in micrometers(μm). It estimates the intensity of the focused laser beam whendiffraction is accounted for. As shown in FIG. 50, the 0.5 mm decenteredlaser beam and the ideal condition are similar in terms of intensitydistribution as the intensity of the focused beam predominantly lies ina tight spot in both the cases. However, in the case of the laser beambeing tilted with respect to the optical axis of the lens, the surfacecontour shows a distribution of intensity forming the shape of a teardrop, FIG. 50 (C), which resembles the cat's paw structure formed due tomultiple fragmentations observed in nanoholes in FIG. 46. Based on theseresults, an angle as small as 0.5° between the incident laser beam axisand the optical axis of the aspheric lens can lead to comatic aberrationin the focal region of the laser beam. Furthermore, the comaticaberration in the focal region of the laser beam can lead to multiplefragmentations of the intensity distribution resulting in the formationof cat's paw structure.

Optical modeling shows that aspheric lens machining of fused silica isinfluenced by tilt between the optical axis of the lens and the laserbeam propagation axis. Thus, it is beneficial to remove angulardeviation between the laser beam axis and the optical axis of theaspheric lens to conclusively study the role of spherical aberration onnanohole formation. In order to obtain beam alignment to reduce comaticaberration, a platform that allows the manipulation of sphericalaberration in the focal region of the laser beam is presented. A set ofthree lenses, two identical plano-concave (f=−100 mm) and one biconvex(f=100 mm), referred to as zoom lens from this point onwards, can beplaced before the aspheric lens in the machining platform. Thearrangement of the zoom lens and the substrate stage is illustrates inFIG. 51.

Software can be used to model the zoom lens to find differentconfigurations, i.e., lens separations, for which spherical aberrationcan be either minimum or significant in the focal region of the laserbeam. Conditions for both positive and negative longitudinal sphericalaberration can be determined using the software. Configurations obtainedby modeling, i.e., with and without spherical aberration, can besubsequently used for laser machining of fused silica substrates. Fusedsilica substrates thus machined can be characterized by SEM analysis ofthe laser damaged regions and the respective cellulose acetate replicasto determine the size of the nanoholes. Results obtained can be comparedand analyzed to understand the role of spherical aberration on theformation of high aspect ratio deep nanoholes.

This system consists of a 527 nm Q-switched Nd:YLF pump laser(Evolution, Coherent, Inc.), a titanium-sapphire laser oscillator(Micra, Coherent, Inc.), and a titanium-sapphire regenerative amplifier(Legend, Coherent, Inc.). The output from the amplifier is a train of upto 1 kHz laser pulses of wavelength 800 nm; but the laser is operated togive a single pulse on-demand. The pulse duration is 40 femtosecondswith maximum energy per pulse of 1.6 mJ. The LabVIEW™ program alsogenerates a trigger pulse (from a DAQ card) to command the laser to givea single pulse when needed.

In order to understand the role of spherical aberration on nanoholeformation, it is informative to compare nanoholes made with a laser beamexhibiting no longitudinal spherical aberration (LSA) and one exhibitingsignificant LSA in its focal region, while at the same time taking careto maintain the same numerical aperture and to avoid overfilling of thefocusing lens, which leads to energy deposited around the focal pointdue to diffraction. A zoom lens consisting of three spherical lenses canbe used to obtain minimum LSA and significant LSA in the focal region ofthe laser beam focused through an aspheric lens. There are two signs ofLSA: negative LSA and positive LSA. Negative LSA (also calledovercorrected spherical aberration) results when the marginal raysrefract less than the paraxial rays, whereas positive LSA(undercorrected) results when the marginal rays refract more than theparaxial rays. Changing the degree of collimation of the beam thatenters the aspheric focusing lens can introduce a significant amount ofLSA in the focal region. A converging laser beam focused through theaspheric lens (but fully filling the entrance pupil) results in positiveLSA, whereas a diverging laser beam results in negative LSA. Negativeand positive LSAs can be introduced in the focal region of the laserbeam by varying the amount of collimation.

The lens separations in the zoom lens required to obtain these threeconditions (positive LSA, minimum LSA, and negative LSA) can be foundwith the help of software modeling. In the model, the lens separationscan be optimized so that the aspheric lens pupil is fully filled and soas to give the greatest divergence, maximum collimation, and greatestconvergence of the beam entering the aspheric lens, which in turnresults in most positive LSA, close to zero LSA, and most negative LSAin the focal region. The diameter of the laser pulse (1/e² value) andthe beam divergence required for modeling were measured to be 6.44 mmand −0.122 milliradians, respectively, by using a power ratio method.The zoom lens can decrease the beam size to exactly fill the pupil ofthe asphere, without overfilling it, and it also can adjust thecollimation of the beam before the asphere and hence the longitudinalspherical aberration in the focal region of the laser pulse. Since thezoom lens can be mounted on a rail of fixed length, the maximumachievable positive and negative LSAs are limited. The three conditionsfound by modeling used for aspheric lens machining experiments are:negative LSA of −49 μm, close to zero LSA (termed best collimation) of+2 μm, and positive LSA of +27 μm. FIG. 53 shows a ray tracing of thesethree conditions and the corresponding Huygens point spread function. Itshows ray tracing of 800 nm wavelength laser beam entering the asphericlens after going through the zoom lens (bottom). The Huygens PSF isplotted at the focal point from the front of the lens on the opticalaxis for the three machining conditions. The distance from the front ofthe lens to the focal position for each of these conditions are: 1.9 mmfor negative LSA, 1.76 mm for best collimation, and 1.711 mm forpositive LSA.

The aspheric lens machining experiments under these three conditions(negative LSA, best collimation, and positive LSA) can be done at threedifferent values of laser pulse energy (1.2 μJ, 3.5 μJ, and 38 μJ) afteraligning the optical elements and the substrate. In order to reducecomatic aberration arising due to angular deviation between the laserbeam path and the aspheric lens optical axis, the three lenses of thezoom lens and the aspheric lens can be aligned with the help of a greenlaser pointer (532 nm wavelength). The green laser is in the visiblerange and is reflected by the aspheric lens coated with ananti-reflective coating for the 600 nm-1050 nm wavelength range.Furthermore, the optical leveling of the fused silica substrate also canbe performed with the green laser beam in lieu of the stylus, so thatsubstantially identical holes are machined for analysis. Note that thecalibration of the surface profile with the stylus prior to femtosecondmachining provides the advantage that the focal position of the samplecan be adjusted to compensate for surface irregularities in thesubstrate. SEM images of the cellulose acetate replicas of the asphericlens machined nanoholes are shown in FIG. 55.

SEM images of the CA replicas show that the aspheric lens machining offused silica with negative LSA did not usually produce deep nanoholesfor the three pulse energies tested. However, some of the nanoholes madewith 38 μJ appear to be ˜8 μm deep from their CA replicas. Note, withrespect to the nanoholes made with negative LSA, the appearance ofconcentric rings in the laser damaged region (or the laser footprint) asseen from their CA replicas. The concentric rings appear to be due todiffraction of the laser pulse and can be seen in the Huygens pointspread function (PSF) in FIG. 53 for negative LSA.

It can be deduced from the SEM images, shown in FIG. 55, that thenanoholes produced in best collimation condition (+2 μm LSA) are deeperthan the expected depth of focus of the tightly focused laser beam andare also deeper than the shallow crater that is usually observed from aself-limiting over-dense laser-induced plasma. The average length of theCA nanowires imprinted from these nanoholes is 10 μm for all the valuesof laser pulse energy, thus the nanoholes are at least up to 10 μm indepth.

It can be seen from the SEM images that even deeper nanoholes areproduced with positive LSA. For LSA of +27 μm and pulse energy of 3.5μJ, the length of the CA nanowires can be measured to be ˜32 μm, whichindicates that the nanoholes can be at least as deep as 32 μm.Furthermore, the nanoholes made at 1.2 μJ pulse energy with the positiveLSA of +27 μm also appear to be around 30 μm deep as indicated by theircellulose acetate replicas. However, the nanoholes made by 38 μJ pulseenergy are only ˜8 μm in depth. The size of the laser footprint, asdepicted by the CA replica, can increase with laser pulse energyirrespective of the sign and the magnitude of LSA. The depth of thenanohole, however, was not found to increase with the laser pulseenergy. In fact, the deepest nanoholes were machined with 1.2 μJ (30 μmdeep) and 3.5 μJ (32 μm deep) laser pulse energy values compared to 38μJ (8 μm deep) in positive LSA conditions.

The formation of 10 μm deep nanoholes in fused silica with reducedspherical aberration in the focal region (best collimation condition,LSA +2 μm) indicates that spherical aberration is not required to createholes deeper than the depth of focus of the beam. For adiffraction-limited focused Gaussian beam, in the paraxial approximation(NA=sin θ≈θ (radians)), the depth of focus is two times the Rayleighrange, 2z_(R)=2λ/(nπNA²), where z_(R) is the Rayleigh range, λ=0.8 μm isthe laser wavelength, n=1.47 is the refractive index of fused silica,and NA is the numerical aperture of the focusing lens. For the asphericlens with NA=0.68, this gives 2z_(R)=0.75 μm. However, the plasma may becreated over a greater depth provided the laser irradiance is above thedamage threshold, which is about 10¹³ W/cm² for fused silica. FIG. 56shows the irradiance of the femtosecond laser pulse as a function ofaxial position around the focal region for the best collimationcondition (focus at 1.76 mm) in air computed using software modeling fora pulse energy of 1.2 μJ. The depth of damage threshold (ΔZ) was foundto be >19 μm. The damage threshold for fused silica is Ith=1013 W/cm2.It can be seen that the depth over which the irradiance is above thedamage threshold is more than 19 μm. This depth of damage threshold (ΔZ)is adequate to account for the observed nanohole depths (10 μm) made byfocusing laser pulses with essentially no spherical aberration if thelaser pulse energy can propagate through the plasma. While only ashallow feature might be expected because laser-induced plasmas arereflective if overdense (when the electron density is high enough suchthat the plasma frequency is above the optical frequency), in the caseof breakdown induced by a femtosecond laser pulse, deeper features canbe produced if a sufficient amount of the pulse energy propagates beforethe plasma is created.

When significant positive spherical aberration is introduced in thefocal region of the laser pulse (LSA of +27 μm), the depth of thenanoholes can be up to 32 μm, which is much deeper than those made withno or negative spherical aberration. The increase in the nanohole depthoccurs even though the calculated depth of damage threshold is less thanthat for no spherical aberration. When greater negative sphericalaberration is introduced (LSA of −49 μm), the nanoholes can be veryshallow at pulse energies of 1.2 and 3.5 μJ and up to about 8 μm for 38μJ. As seen in FIG. 57 and FIG. 58, the depth of damage threshold in airalong the axis was calculated to be ˜14 μm for LSA of +27 μm and ˜15 μmfor LSA of −49 μm. The depth of damage threshold (ΔZ) was found to be˜14 μm. However, it should be noted that, refraction of the beam as itenters the surface induces spherical aberration near the focus that isnot accounted for above. It is possible that this surface-inducedaberration will further extend the depth of damage threshold along theaxis to enable >30 μm deep nanoholes to be created in fused silica forthe case of initial positive spherical aberration. In addition, theabove calculations do not account for non-linear effects, such asKerr-induced self-focusing and/or possible beam front reshaping into adiffraction-free Bessel beam at the surface of fused silica. Sucheffects may contribute as mechanisms for deep-hole formation. Insummary, spherical aberration can alter the depths of the created holes,with positive LSA creating deeper holes than no or negative LSA.

Single-pulse amplified femtosecond laser machining has been developedinto a new nanofabrication technology for production of templates ontransparent substrates. It is essentially a direct-write nanolithographytechnique, which can pattern the surface of wide band gap materials likefused silica with user-defined arrangements of nanoholes with aspectratios >10:1 and depths >10 μm. The lateral and vertical dimensions ofthe nanoholes are shown to be controllable during laser machining byregulating processing conditions and by chemical etching post-machining.The process is shown to be simple, economical, and less time consumingthan other processes, thus making it useful for large scale productionof templates.

Nanoholes made by amplified femtosecond laser single pulses, e.g., ofenergy 5.2 μJ, pulse duration 160 femtosecond, and wavelength 780 nmfocused through a microscope objective lens of numerical apertureNA=1.25 in air, can be at least as deep as 14 βμm by cellulose acetatereplication of the patterned fused silica surface.

The diameter and the depth of the nanoholes can be affected by therelative position of the optical focus with respect to the substratesurface during laser machining. The deepest nanoholes can form when theoptical focus is within the bulk of the substrate at a certain locationfrom the substrate surface. Furthermore, the size and the morphology ofthe nanoholes made on fused silica substrates can be modified by acombination of sequential etching in hydrofluoric acid and potassiumhydroxide.

The templates made by single-pulse amplified femtosecond laser machiningcan be useful as molds for imprinting high aspect ratio polymerstructures by replication procedures. Two replication methods forimprinting polymer structures are shown to be beneficial: film basedreplication and solution casting. The film based replication procedurecan use commercially available polymer films, whereas solution castingcan be based on polymer solutions made in common organic solvents.Solution casting presents several advantages over film-basedreplication. Solution casting offers a measure of control over the finalchemistry of the imprinted polymer structures, and can allow imprintingof thermoset polymers, which are otherwise not possible with film-basedreplication. Nanocones and micropillars of polycaprolactone (PCL),PCL—polyethylene glycol (PEG), cellulose acetate (CA), polyethylene(PE), polyvinyl alcohol (PVA), polymethylmethacrylate (PMMA), Collodion,and polydimethylsiloxane (PDMS) can be imprinted from as-machined andetched fused silica templates, respectively, by solution casting. PCLand PCL-PEG structures imprinted from as-machined templates can stretchduring imprint lift-off and take the form of nanocarpets or nanomatswith very high surface area and aspect ratios greater than 200:1. Allthe aforementioned polymers except PCL and PCL-PEG can adopt the shapeand dimensions close to that of the nanoholes they are imprinted from,though some stretching of the nanocones is also observed. Furthermore,most of the PDMS nanocones can fall over, but without breaking orstretching, because of the low Young's modulus of PDMS. None of thepolymer micropillars have been found to stretch, break, or fall over dueto stiffness.

Polymer structures imprinted from amplified femtosecond laser machinedfused silica substrates can be functionalized by coating with thinlayers of gold, platinum, and palladium by sputter coating and silica bychemical vapor deposition. Sputter coating of noble metals can beperformed at cryogenic temperatures to mitigate bending or collapsing ofpolymer nanowires by impact of fast moving ions. For this purpose, thesputtering system can include a liquid nitrogen cold finger so that thepolymer nanowires become stiff below their glass transition temperaturesand withstand the impact of ions during coating deposition. Alow-temperature chemical vapor deposition process can deposit silica oncellulose acetate nanowires to fabricate silica nanowires, based on thehydrolysis of silicon tetrachloride molecules adsorbed at the surface ofCA nanowires, which completes in two separate beakers and results in theformation of a thin layer of silica. The thickness of the silica coatingcan increase with the increase in the exposure time to silicontetrachloride.

Silica nanowires produced by this technique can be useful as syntheticcell culture substrates with tunable 3D microenvironment for cellbehavioral studies. The cells can adhere and align along silicananowires, thus making silica nanowires a useful material for tissueengineering. Furthermore, focused ion beam sectioning of a silicananowire tip demonstrates the utility of fabricating open-tip silicananoneedles that can find applications as cellular patch clamps forintravital imaging and electrophysiological studies.

The role of spherical aberration on the formation of high aspect ratioand deep nanoholes was investigated with the help of aspheric lensmachining and optical modeling using Zemax software. An aspheric lens ofNA=0.68 can be used to machine nanoholes at the surface of fused silicaby varying the amount of longitudinal spherical aberration (LSA) in thefocal region of the laser pulse determined by optical modeling. NegativeLSA, minimum possible LSA, and positive LSA can be introduced in thefocal region of a 40 femtosecond laser pulse of wavelength 800 nm byvarying the collimation at the entrance pupil of the aspheric lens. Thedepths of the nanoholes thus produced can be determined by celluloseacetate replication, and it the deepest nanoholes can be made bypositive LSA, the next deepest by minimum LSA and the shallowest bynegative LSA. The nanoholes made by minimum LSA of +2 μm can be 10 μmdeep for pulse energy in the range 1.2-38 μJ, which indicates thatspherical aberration is not required to create holes deeper than thedepth of focus of the beam. However, if sufficient pulse energy can passthrough or around the plasma, the calculated depth of damage thresholdalong the axis is adequate to account for the observed hole depths. Whenpositive longitudinal spherical aberration of 27 μm is introduced in thefocal region of the femtosecond laser pulse, the depth of the nanoholescan increase to 30-32 μm for pulse energies of 1.2-3.5 μJ, even thoughthe calculated depth of damage threshold decreases to 14 μm.

The formation of deep and high aspect ratio nanoholes cannot beexclusively attributed to the presence of spherical aberration in thefocal region of the laser pulse, though hole-depth can increase with theincrease in positive longitudinal spherical aberration up to a certainpulse energy. Transmission of a significant part of the pulse energythrough or around the first part of the laser-induced plasma as well asself-focusing due to Kerr nonlinearity and/or nonlinear laser pulsereshaping into a non-diffractive Bessel beam could also be important inthe formation of deep and high aspect ratio nanoholes at the surface offused silica by single-pulse femtosecond laser ablation.

Although specific example embodiments of the present technology havebeen described above in detail, the description is for purposes ofillustrating the range of embodiments of the technology conceived by theinventors. Various modifications of, and equivalent elementscorresponding to, the disclosed aspects of the technology, in additionto those described above, can be made by those having ordinary skill inthe art without departing from the spirit and scope of the followingclaims, the scope of which is to be accorded an interpretation toencompass such modifications and equivalent structures.

1-16. (canceled)
 17. A nanostructure, comprising: (a) a polymer, and (b)an aspect ratio of at least ten-to-one, wherein said nanostructure isprepared from a nanohole formed within a transparent substrate byablation using a single femtosecond laser pulse.
 18. The nanostructureof claim 17, wherein said aspect ratio exceeds twenty-five-to-one. 19.The nanostructure of claim 17, wherein said nanostructure comprises alength greater than ten microns and a width less than one micron. 20.The nanostructure of claim 19, wherein said nanostructure comprises alength of at least thirty microns and a width less than one micron. 21.The nanostructure of claim 17, wherein said nanostructure is cast,synthesized, molded, replicated, imprinted, or embossed from saidnanohole.
 22. The nanostructure of claim 17, further comprising an arrayof nanostructures, each having an aspect ratio of at least ten-to-one,said array being fabricated from a plurality of nanoholes, each formedwithin a transparent substrate by ablation using a single femtosecondlaser pulse.
 23. The nanostructure of claim 22, wherein saidnanostructures are non-randomly positioned.
 24. A method for fabricatinga nanostructure, comprising: (a) providing a template of transparentmaterial; (b) ablating at least one nanohole in said template using asingle femtosecond laser pulse; (c) filling said at least one nanoholewith a first polymeric material; and (d) extracting said first polymericmaterial as the nanostructure, wherein said nanostructure has an aspectratio of at least ten-to-one.
 25. The method of claim 24, wherein saidfilling comprises filling a plurality of non-randomly positionednanoholes in said template with a first polymeric material to replicatean array of non-randomly positioned nanostructures.
 26. The method ofclaim 24, further comprising treating a surface of the template with amaterial to modify the surface and bonding characteristics associatedwith said template and said first polymeric material.
 27. The method ofclaim 24, wherein said ablating comprises introducing a generallyspherical aberration into a focus associated with the laser pulse todistribute the focused light and increase an aspect ratio associatedwith said at least one nanohole.
 28. The method of claim 24, whereinsaid ablating comprises introducing an aberration of a laser beam toalter a shape of the focused beam and the shape or number of said atleast one nanohole.
 29. The method of claim 24, further comprisingmodifying the template to enlarge or change a geometry of said at leastone nanohole.
 30. The method of claim 29, wherein said modifyingcomprises using chemical reactions, vapor, etching, or liquid growthprocesses.
 31. A reusable template for high-throughput production ofnanostructure arrays, comprising a transparent material and a pluralityof high aspect ratio nanoholes ablated in a surface thereof by aplurality of single femtosecond laser pulses.
 32. The template of claim31, wherein said nanoholes are non-randomly positioned.
 33. The templateof claim 31, wherein said aspect ratio is at least ten-to-one.
 34. Thetemplate of claim 31, wherein said nanoholes are at least ten micronsdeep.
 35. The template of claim 31, wherein said transparent materialcomprises: (a) any glass or amorphous material, (b) any thermosetting orthermoplastic polymer, or (c) any crystalline material.
 36. The templateof claim 35, wherein said glass or amorphous material comprises fusedsilica, silica glasses, quartz, soda-lime-silica glass, BK7 opticalglass or other borosilicate.
 37. The template of claim 35, wherein saidthermosetting or thermoplastic polymer comprises polycarbonate, acrylic,polymethylmethacralate, polydimethylsiloxane, or polyvinal alcohol. 38.The template of claim 35, wherein said crystalline material comprisesdiamond or sapphire.
 39. The template of claim 31, wherein saidtransparent material is fused silica.
 40. A nanostructure array,comprising a plurality of high aspect ratio polymer nanostructurespositioned in a user-defined pattern and fabricated from a plurality ofnanoholes formed within a transparent substrate by ablation using anequivalent number of femtosecond laser pulses.
 41. The array of claim40, wherein said nanoholes have a depth to width ratio of at least 10:1.42. The array of claim 41, wherein said nanoholes have a width of 1 μmor less.
 43. The array of claim 41, wherein said nanoholes have a depththat is at least twenty-five times greater than said width.